Polyvalent cation-sensing receptor in atlantic salmon

ABSTRACT

The present invention encompasses three full length nucleic acid and amino acid sequences for PolyValent Cation-Sensing Receptors (PVCR) in Atlantic Salmon. These PVCR have been named SalmoKCaR#1, SalmoKCaR#2, and SalmoKCaR#3. The present invention includes homologs thereof, antibodies thereto, and methods for assessing SalmoKCaR nucleic acid molecules and polypeptides. The present invention further includes plasmids, vectors, host cells containing the nucleic acid sequences of SalmoKCaR#1,2 and/or 3.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 10/125,772, filed Apr. 18,2002, which is a continuation-in-part of U.S. application Ser. No.10/121,441, filed Apr. 11, 2002, now abandoned, which is acontinuation-in-part of International Application No. PCT/US01/31704(WO02/031149), which designated the United States, filed Oct. 11, 2001,now abandoned, which claims the benefit of U.S. Provisional ApplicationNo. 60/240,392, filed on Oct. 12, 2000, and U.S. Provisional ApplicationNo. 60/240,003, filed on Oct. 12, 2000. The entire teachings of theabove applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

In nature, anadromous fish like salmon live most of their adulthood inseawater, but swim upstream to freshwater for the purpose of breeding.As a result, anadromous fish hatch from their eggs and are born infreshwater. As these fish grow, they swim downstream and gradually adaptto the seawater.

Currently, wild Atlantic salmon are classified as endangered species inmultiple areas of their native habitats. Among the reasons for theirdecline has been man made alterations in freshwater conditions in theirnative streams that have produced multiple problems with theirmigration, spawning, smoltification and survival. One problemcomplicating the effective restoration of wild Atlantic salmon is thelack of a fundamental understanding of how these deleteriousenvironmental conditions effect the salmon's ability to home tofreshwater streams from the ocean, interchangeably adapt to freshwaterand seawater as well as feed and grow in both salinity environments.

Despite the decline of wild populations, the global aquaculture industryhas utilized Atlantic salmon as one of chief fish species forlarge-scale marine farming operations. At the present time, large scalebreeding programs of Atlantic salmon provide for high quality fish usedin production by selection of specific traits among them rapid growth,seawater adaptability, flesh quality and taste.

However, fish hatcheries have experienced some difficulty in raisingsalmon because the window of time in which the pre-adult salmon adaptsto seawater (e.g., undergoes smoltification) is short-lived, and can bedifficult to pinpoint. As a result, these hatcheries can experiencesignificant morbidity and mortality when transferring salmon fromfreshwater to seawater. Additionally, many of the salmon that do survivethe transfer from freshwater to seawater are stressed, and consequently,experience decreased feeding, and increased susceptibility to disease.Therefore, salmon often do not grow well after they are transferred toseawater.

The aquaculture industry loses millions of dollars each year due toproblems it encounters in transferring salmon from freshwater toseawater. Therefore, a need exists to gain a better understanding of thebiological processes of salmon that are related to smoltification andadaptation to varying salinities, including seawater. In particular, aneed exists to identify genes that play an important role in theseareas.

SUMMARY OF THE INVENTION

The present invention relates to genes that allow fish to sense andadapt to ion concentrations in the surrounding environment. Modulatingone or more of these genes allow anadromous fish like salmon to betteradapt to seawater during smoltification, which in turn allows salmon togrow faster and stronger after transfer to seawater. A gene, called aPolyValent Cation-sensing Receptor (PVCR), has been isolated in severalspecies of fish, and in particular, in Atlantic Salmon. In fact, threeforms of the PVCR have been isolated in Atlantic Salmon, and have beentermed, “SalmoKCaR” genes and individually referred to as “SalmoKCaR#1”,“SalmoKCaR#2” and “SalmoKCaR#3.” “PVCR” and “SalmoKCaR” are usedinterchangeably when referring to Atlantic Salmon. These three geneswork together to alter the salmon's sensitivity to the surrounding ionconcentrations, as further described herein.

The invention embodies nucleic acid molecules (e.g., RNA, genomic DNAand cDNA) having nucleic acid sequences of SalmoKCaR#1 (SEQ ID NO: 7),SalmoKCaR#2 (SEQ ID NO: 9), or SalmoKCaR#3 (SEQ ID NO: 11). Theinvention also embodies polypeptide molecules having amino acidsequences of SalmoKCaR#1 (SEQ ID NO: 8), SalmoKCaR#2 (SEQ ID NO: 10), orSalmoKCaR#3 (SEQ ID NO: 12). The present invention, in particular,encompasses isolated nucleic acid molecules having nucleic acidsequences of SEQ ID NO: 7, 9, or 11; the complementary strand thereof;the coding region of SEQ ID NO: 7, 9, or 11; or the complementary strandthereof. The present invention also embodies nucleic acid molecules thatencode polypeptides having an amino acid sequence of SEQ ID NO: 8, 10,or 12. The present invention, in another embodiment, includes isolatedpolypeptide molecules having amino acid sequences that comprise SEQ IDNO: 8, 10, or 12; or amino acid sequences encoded by the nucleic acidsequence of SEQ ID NO: 7, 9, or 11.

In one embodiment, the present invention pertains to isolated nucleicacid molecules that have a nucleic acid sequence with at least about 70%(e.g., 75%, 80%, 85%, 90%, or 95%) identity with SEQ ID NO: 7, 9, or 11,or the coding region of SEQ ID NO: 7, 9, or 11. Such a nucleic acidsequence encodes a polypeptide that allows for or assists in one or moreof the following functions: sensing at least one SalmoKCaR modulator inserum or in the surrounding environment; adapting to at least oneSalmoKCaR modulator present in the serum or surrounding environment;imprinting Atlantic Salmon with an odorant; altering water intake;altering water absorption; or altering urine output.

The present invention further includes nucleic acid molecules thathybridize with SalmoKCaR#1, SalmoKCaR#2, or SalmoKCaR#3, but not to theShark Kidney Calcium Receptor Related Protein (SKCaR) nucleic acidsequence. SKCaR is a PVCR isolated from dogfish shark. Specifically, thepresent invention relates to an isolated nucleic acid molecule thatcontains a nucleic acid sequence that hybridizes under high stringencyconditions to SEQ ID NO: 7, 9, or 11; or the coding region of SEQ ID NO:7, 9, or 11; but excluding those that hybridize to SEQ ID NO: 1 underthe same conditions.

The present invention also includes probes, vectors, viruses, plasmids,and host cells that contain the nucleic acid sequences, as describedherein. In particular, the present invention includes probes (e.g.,nucleic acid probes or DNA probes) having a sequence from SEQ ID NO: 7,but not SEQ ID NO: 1. The present invention encompasses nucleic acid orpeptide molecules purified or obtained from clones deposited withAmerican Type Culture Collection (ATCC), Accession No: PTA-4190,PTA-4191, or PTA-4192.

In another embodiment, the present invention includes isolatedpolypeptide molecules having at least about 70% (e.g., 75%, 80%, 85%,90%, or 95%) identity with SEQ ID NO: 8, 10, or 12; or an amino acidsequence encoded by the nucleic acid sequence of SEQ ID NO: 7, 9, or 11.These polypeptide molecules have one or more of the following functions:sensing at least one SalmoKCaR modulator in serum or in the surroundingenvironment; adapting to at least one SalmoKCaR modulator present in theserum or surrounding environment; imprinting Atlantic Salmon with anodorant; altering water intake; altering water absorption; or alteringurine output.

Additionally, the present invention relates to antibodies thatspecifically bind to or are produced in reaction to polypeptidemolecules described herein. The invention further includes fusionproteins that contain one of the polypeptide molecules described herein,and a portion of an immunoglobulin.

The present invention also pertains to assays for determining thepresence or absence of a SalmoKCaR in a sample by contacting the sampleto be tested with an antibody specific to at least a portion of theSalmoKCaR polypeptide sufficiently to allow formation of a complexbetween SalmoKCaR and the antibody, and detecting the presence orabsence of the complex formation. Another assay for determining thepresence or absence of a nucleic acid molecule that encodes SalmoKCaR ina sample involves contacting the sample to be tested with a nucleic acidprobe that hybridizes under high stringency conditions to a nucleic acidmolecule having a sequence of SEQ ID NO: 7, 9, or 11, sufficiently toallow hybridization between the sample and the probe; and detecting theSalmoKCaR nucleic acid molecule in the sample. Such assay methods alsoinclude methods for determining whether a compound is a modulator ofSalmoKCaR. These methods include contacting a compound to be tested witha cell that contains SalmoKCaR nucleic acid molecules and/or expressesSalmoKCaR proteins, and determining whether compounds are modulators bymeasuring the expression level or activity (e.g., phosphorylation,dimerization, proteolysis or intracellular signal transduction) ofSalmoKCaR proteins. In one embodiment, one can measure changes thatoccur in one or more intracellular signal transduction systems that arealtered by activation of the expressed proteins coded for by a single orcombination of nucleic acids. Such methods can also encompass contactinga compound to be tested with a cell that comprises one or more ofSalmoKCaR nucleic acid molecules; and determining the level ofexpression of said nucleic acid molecule. An increase or decrease in theexpression level, as compared to a control, indicates that the compoundis a modulator.

Lastly, the present invention relates to transgenic fish encoding aSalmoKCaR polypeptide or having one or more nucleic acid molecules thatcontain the SalmoKCaR nucleic acid sequence, as described herein.

The present invention allows for a number of advantages, including theability to more efficiently grow Atlantic Salmon, and in particular,transfer them to seawater with increased growth and reduce mortality.The technology of the present invention also allows for assaying ortesting these salmon to determine if they are ready for transfer toseawater, so that they can be transferred at the optimal time. Thetechnology of the present invention provides for the imprinting ofsalmon with an odorant so that the salmon, once imprinted, can latermore easily recognize and/or distinguish the odorant. For example, anattractant that has been used to imprint salmon can be added to feed sothat the salmon will consume more feed and grow at a faster rate. Anumber of additional advantages for the present invention exist and areapparent from the description provided herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-E show the annotated nucleotide sequence (SEQ ID NO: 1) and thededuced amino acids sequence (SEQ ID NO: 2) of SKCaR with the OpenReading Frame (ORF) starting at nucleotide (nt) 439 and ending at 3516.

FIG. 2 is a graphical representation showing a normalized calciumresponse (%) against the amount of Calcium (mM) of the SKCaR-I proteinwhen modulated by alternations in extracellular NaCl concentrations.

FIG. 3 is a graphical representation showing a normalized calciumresponse (%) against the amount of magnesium (mM) of the SKCaR proteinin increasing amounts of extracellular NaCl concentrations.

FIG. 4 is a graphical representation showing the EC50 for calciumactivation of shark CaR (mM) against the amount of sodium (mM) of theSKCaR-I protein in increasing amounts of extracellular NaClconcentrations.

FIG. 5 is a graphical representation showing the EC50 for magnesiumactivation of shark CaR (mM) against the amount of sodium (mM) of theSKCaR protein in increasing amounts of extracellular NaClconcentrations.

FIG. 6 is a graphical representation showing the EC50 for magnesiumactivation of shark CaR (mM) against the amount of sodium (mM) of theSKCaR protein in increasing amounts of extracellular NaCl concentrationsand added amounts of calcium (3mM).

FIGS. 7A and 7B show an annotated partial nucleotide sequence (SEQ IDNO: 3) and the deduced amino acids sequence (SEQ ID NO: 4) of anAtlantic salmon polyvalent cation-sensing receptor protein.

FIGS. 8A-8C show a second annotated partial nucleotide sequence (SEQ IDNO: 5) and the deduced amino acids sequence (SEQ ID NO: 6) of anAtlantic salmon polyvalent cation-sensing receptor protein.

FIGS. 9A-E show the nucleic acid (SEQ ID NO: 7) and amino acid (SEQ IDNO: 8) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#1 withthe ORF starting at nt 180 and ending at 3005.

FIGS. 10A-E show the nucleic acid (SEQ ID NO: 9) and amino acid (SEQ IDNO: 10) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#2with the ORF starting at nt 270 and ending at 3095.

FIGS. 11A-D show the nucleic acid (SEQ ID NO: 11) and amino acid (SEQ IDNO: 12) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#3with the ORF starting at nt 181 and ending at 2733.

FIGS. 12A-L are an alignment showing nucleic acid sequences of twopartial Atlantic Salmon Clones (SEQ ID NO: 3 and 5), SalmoKCaR#1 (SEQ IDNO: 7), SalmoKCaR#2 (SEQ ID NO: 9), and SalmoKCaR#3 (SEQ ID NO: 11).

FIGS. 13A-C are an alignment showing amino acid sequences of two partialAtlantic Salmon Clones (SEQ ID NO: 4 and 6), SalmoKCaR#1 (SEQ ID NO: 8),SalmoKCaR#2 (SEQ ID NO: 10), and SalmoKCaR#3 (SEQ ID NO: 12).

FIG. 14 is photograph showing a Southern blot in which SalmoKCaR#1, 2,and 3 hybridize to nucleic acid derived from SKCaR.

FIGS. 1 5A-H are an alignment of the full length nucleic acid sequencesof SalmoKCaR#1, 2, and 3 (SEQ ID NO: 7, 9, and 11, respectively).Alignment obtained using Clustal method with weighted residue weighttable.

FIGS. 16A-D are an alignment of the full length amino acid sequences ofHuman Parathyroid Calcium Receptor (HuPCaR) (SEQ ID NO: 28), SKCaR (SEQID NO: 2), SalmoKCaR#1 (SEQ ID NO: 8), SalmoKCaR#2 (SEQ ID NO: 10) andSalmoKCaR#3 (SEQ ID NO: 12). Alignment obtained using Clustal methodwith PAM250 residue weight table.

FIGS. 17A-F are graphical representations comparing six photographs ofReverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis offreshwater (FIGS. 17B, D and F) and seawater (FIGS. 17A, C and E)adapted Atlantic salmon tissues (gill, nasal lamellae, urinary bladder,kidney, stomach, pyloric caeca, proximal intestine, distal intestine,brain, pituitary gland, olfactory bulb, liver and muscle) using eitherdegenerate PVCR (FIGS. 17A-D) or salmon actin PCR primers (FIGS. 17E,F).Wells 1-14 for FIGS. 17A-F, top row, are designated as follows: ladder,gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca,proximal intestine, distal intestine, brain, pituitary gland, olfactorybulb, liver and muscle, respectively. Wells 1, 2, 7, 9, and 12, bottomrow, for FIGS. 17A, C, and E are designated as ladder, water, SalmoKCaR#1, SalmoKCaR#2 and SalmoKCaR#3, respectively, and wells 1, 2, 3, 7, 9,and 12, bottom row, for FIGS. 17B,D, and F are designated as ladder,water, ovary, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively.

FIG. 18A is photograph of a RT-PCR analysis using degenerate primers ofsteady state SalmoKCaR mRNA transcripts from kidney tissue of AtlanticSalmon adapted to freshwater, after 9 weeks of Process II treatment or26 days after transfer to seawater. Process II treatment is defined inthe Exemplification.

FIG. 18B is a photograph of a RT-PCR analysis showing increased steadystate expression of SalmoKCaR transcripts in pyloric caeca of Process IItreated and seawater fish as compared to freshwater Atlantic salmonsmolt. Using degenerate (SEQ ID Nos 13 and 14) or actin (SEQ ID No 22and 23) primers, samples of either freshwater (Panel A Lanes 3 and 6),Process II treated (Panel A Lanes 4 and 7) or seawater adapted (Panel ALanes 5 and 8) Atlantic salmon smolt were analyzed by RT-PCR. To controlfor differences in sample loading, these identical samples weresubjected to PCR analysis using actin specific primer (Panel A, Lanes3-5). Note that both ethidium bromide stained gel (Panel A) and itscorresponding Southern blot (Panel C) show increased amounts ofSalmoKCaR transcripts in pyloric caeca from Process II and seawateradapted fish as compared to freshwater. As a control, Panel Bdemonstrates that these degenerate primers amplify SalmoKCaR #1 (Lane1), SalmoKCaR #2 (Lane 2) and SalmoKCaR #3 (Lane 3) transcripts.

FIG. 18C is a photograph of RT-PCR analysis showing expression ofSalmoKCaR transcripts in various stages of Atlantic salmon embryodevelopment. Using degenerate (SEQ ID Nos. 13 and 14) or actin (SEQ IDNo 22 and 23) primers, RNA obtained from samples of whole Atlanticsalmon embryos at various stages of development were analyzed forexpression of SalmoKCaRs using RT-PCR. Ethidium bromide staining ofsamples from dechorionated embryos (Lane 1), 50% hatched (Lane 2), 100%hatched (Lane 3), 2 weeks post hatched (Lane 4) and 4 weeks post hatched(Lane 5) shows that SalmoKCaR transcripts are present in Lanes 1-4).Southern blotting of the same gel (Panel C) confirms expression ofSalmoKCaRs in embryos from very early stages up to 2 weeks afterhatching. No expression of SalmoKCaR was observed in embryos 4 weeksafter hatching. Panel B shows the series of controls where PCRamplification of actin content of each of the 5 samples shows they areapproximately equal (lanes 1-5).

FIG. 19 is a photograph of a RNA blot containing 5 micrograms of poly A⁺RNA from kidney tissue dissected from either freshwater adapted (FW) orseawater adapted (SW) Atlantic salmon probed with full length SalmoKCaR#1 clone.

FIGS. 20A-F are graphical representations comparing six photographsshowing RT-PCR analysis of freshwater (FIGS. 20B, D and F) and seawater(FIGS. 20A, C and E) adapted Atlantic salmon tissues using eitherSalmoKCaR #3 specific PCR (FIGS. 20A-D) primers or salmon actin PCRprimers (FIGS. 20E,F). Wells 1-14 for FIGS. 20A-F, top row, aredesignated as follows: ladder, gill, nasal lamellae, urinary bladder,kidney, stomach, pyloric caeca, proximal intestine, distal intestine,brain, pituitary gland, olfactory bulb, liver and muscle, respectively.Wells 1, 2, 8, 11, and 14, bottom row, for FIGS. 20A, C, and E aredesignated as ladder, water, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3,respectively, and wells 1, 2, 3, 8, 11, and 14, bottom row, for FIGS.20B,D, and F are designated as ladder, water, ovary, SalmoKCaR #1,SalmoKCaR#2 and SalmoKCaR#3, respectively.

FIGS. 21A-F are graphical representations comparing six photographsshowing RT-PCR analysis of freshwater (FIGS. 21B, D and F) and seawater(FIGS. 21A, C and E) adapted Atlantic salmon tissues using eitherSalmoKCaR #1 specific PCR primers or salmon actin PCR primers. Wells1-14 for FIGS. 21A-F, top row, are designated as follows: ladder, gill,nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca,proximal intestine, distal intestine, brain, pituitary gland, olfactorybulb, liver and muscle, respectively. Wells 1, 2,3, 5, 6, and 7. bottomrow, for FIGS. 21A, C, and E are designated as ladder, water, Kidney-RT,SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively, and wells 1, 2,3, 5, 6, and 7, bottom row, for FIGS. 21B, D, and F are designated asladder, water, ovary, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3,respectively.

FIGS. 22A-F are graphical representations comparing six photographsshowing RT-PCR analysis of freshwater (FIGS. 22B, D and F) and seawater(FIGS. 22A, C and E) adapted Atlantic salmon tissues using eitherSalmoKCaR #2 specific PCR primers (FIGS. 22A-D) or salmon actin PCRprimers (FIGS. 22E,F). Wells 1-14 for FIGS. 22A-F, top row, aredesignated as follows: ladder, gill, nasal lamellae, urinary bladder,kidney, stomach, pyloric caeca, proximal intestine, distal intestine,brain, pituitary gland, olfactory bulb, liver and muscle, respectively.Wells 1, 2,3, 5, 6, and 7. bottom row, for FIGS. 22A, C, and E aredesignated as ladder, water, Kidney-RT, SalmoKCaR #1, SalmoKCaR#2 andSalmoKCaR#3, respectively, and wells 1, 2, 3, 5, 6, and 7, bottom row,for FIGS. 22B, D, and F are designated as ladder, water, ovary,SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively.

FIG. 23 is a schematic diagram illustrating industry practice for salmonaquaculture production, prior to the discovery of the present invention.The diagram depicts key steps in salmon production for S0 (75 gram) andS1 (100 gram) smolts. The wavy symbol indicates freshwater while thebubbles indicate seawater.

FIG. 24A is a graphical representation comparing the weekly feedconsumption on a per fish basis between Process I treated smoltsweighing approximately 76.6 gm vs industry standard smolt weighingapproximately 95.8 gm. These data are derived from individual netpens offish containing about 10,000-50,000 fish per pen. As shown, fish treatedwith Process I consumed approximately twice as much feed per fish duringtheir first week after seawater transfer as compared to the largeindustry standard smolts weekly food consumption after 30 days. ProcessI treatment is defined in the Exemplification.

FIG. 24B is a graphical representation illustrating length (cm) andweight (gm) of Process I Smolts 50 days after ocean netpen placement.Process I smolts had an average weight of 76.6 gram when placed inseawater and were sampled after 50 days.

FIG. 25 is a graphical representation illustrating length (cm) andweight (gm) of representative Process I smolts prior to transfer toseawater.

FIG. 26 is a graphical representation illustrating length (cm) andweight (gm) of Process I smolts before transfer, and mortalities aftertransfer to ocean netpens.

FIG. 27 is a three dimensional graph illustrating the survival over 5days of Arctic Char in seawater after being maintained in freshwater,Process I for 14 days, and Process I for 30 days.

FIG. 28 is a graphical representation illustrating the length (cm) andweight (gm) of St. John/St. John Process II smolts prior to seawatertransfer. Process II is defined in the Exemplification Section.

FIGS. 29A and 29B are graphical representations illustrating weight (gm)and length (cm) of Process II smolt survivors and mortalities 5 daysafter transfer to seawater tanks (A), and 96 hours after transfer toocean netpens (B).

FIGS. 30A-G are photographs of immunocytochemistry of epithelia of theproximal intestine of Atlantic Salmon illustrating SalmoKCaRlocalization and expression.

FIG. 31 is a photograph of a Western Blot of intestinal tissue fromsalmon subjected to Process I for immune (lane marked CaR, e.g., aSalmoKCaR) and preimmune (lane marked preimmune) illustrating SalmoKCaRexpression.

FIGS. 32A-C are photographs of immunolocalization of the SalmoKCaR inthe epidermis of salmon illustrating SalmoKCaR localization andexpression.

FIG. 33 is a graphical representation quantifying the Enzyme-LinkedImmunoSorbent Assay (ELISA) protein (ng) for various tissue samples(e.g., gill, liver, heart, muscle, stomach, olfactory epithelium,kidney, urinary bladder, brain, pituitary gland, olfactory bulb, pyloricceacae, proximal intestine, and distal intestine) from a single fish.

FIG. 34 is a photograph of a RT-PCR amplification of a partial SalmoKCaRmRNA transcript from various tissues (gill, nasal lamellae, urinarybladder, kidney, intestine, stomach, liver, and brain (Wells 1-8,respectively)) of Atlantic Salmon. RT-PCR reactions were separated bygel electrophoresis and either stained in ethidium bromide (EtBr) ortransferred to a membrane and Southern Blotted (SB) using a 32P-labeled653 basepair (bp) genomic DNA fragment from the Atlantic salmonSalmoKCaR gene. Wells 9 and 10 are water (blank) and positive control,respectively.

FIG. 35 is a series of photographs of immunocytochemistry showing theSalmoKCaR localization of Atlantic Salmon Olfactory Bulb Nerve andLamellae using an anti-SalmoKCaR antibody.

FIG. 36 is a schematic illustrating the effect of external and internalionic concentrations on the olfactory lamellae in response to SalmoKCaRmodulators.

FIG. 37A is a photograph of immunocytochemistry showing the SalmoKCaRprotein expression in the developing nasal lamellae (Panel A) andolfactory bulb (Panel B) after hatching of Atlantic salmon using ananti-SalmoKCaR antibody.

FIG. 37B is a photograph of immunocytochemistry of Atlantic salmon ortrout larval fish using Sal-I antiserum shows abundant PVCR proteinexpression by selected cells. Specific binding of Sal-I antiserumdenoting the presence of PVCR protein is shown by the dark reactionproduct. Staining of myosepta between various muscle bundles of larvalfish is shown by asterisks (panel A). Panel B shows the head of a troutlarvae in cross section where abundant PVCR protein is present in theskin (asterisks) and developing nasal lamellae (open arrowhead). Panel Cshows PVCR expression in the developing otolith as well as localizedPVCR protein in epithelial cells immediately adjacent to it. Panels Dand E show high magnification views of myosepta shown in Panel A. Notethe pattern of localized expression of PVCR protein where some cellscontain large amounts of PVCR protein while those immediately adjacentto them have little or no expression. Panel F shows a corresponding H+Esection where myosepta (open arrowheads) can be clearly distinguishedfrom intervening muscle bundles.

FIG. 37C is a photograph showing localization of Sal ADD antiserum byimmunocytochemistry. Panel A shows the pattern of immunostaining ofimmune anti-Sal ADD serum as compared to lack of reactivity displayed bypreimmune anti-Sal ADD serum when exposed to identical kidney tissuesections (Panel B). Note that anti-Sal ADD reactivity (denoted byarrows) is similar if not identical to that displayed by Sal-Iantiserum. Corresponding kidney tubules exposed to preimmune antiserumshow no reactivity (denoted by asterisks).

FIG. 38 is a photograph of immunocytochemistry showing the PVCRlocalization in nasal lamellae of dogfish shark using an anti-PVCRantibody.

FIG. 39 is a photograph of a Southern blot of RT-PCR analyses of tissuesfrom Atlantic Salmon showing the presence of SalmoKCaR mRNA in nasallamellae of freshwater adapted fish. Wells 1-10 are designated asfollows: gill, nasal lamellae, urinary bladder, kidney, intestine,stomach, liver, brain, water (blank) and positive control, respectively.

FIG. 40 is a histogram illustrating the amount of SalmoKCaR protein, asdetermined by an ELISA (ng) for various tissue samples (gill, liver,heart, muscle, stomach, olfactory epithelium, kidney, urinary bladder,brain, pituitary gland, olfactory bulb, pyloric ceacae, proximalintestine, and distal intestine).

FIG. 41 shows the raw and integrated recordings from high resistanceelectrodes of freshwater adapted Atlantic Salmon when exposed to 500 μML-alanine, 1 mmol calcium, 50 μM Gadolinium, and 250 mmol of NaCl. Thefigures show the existence of an olfactory recording in response toL-alanine, calcium, gadolinium, and NaCl.

FIG. 42 is a graph showing the response data for freshwater adaptedAtlantic salmon nasal lamellae for calcium, magnesium, gadolinium, andsodium chloride normalized to the signal obtained with 10 mM Calcium.

FIG. 43 shows raw recording from high resistance electrodes of olfactorynerve impulse in the presence of a repellant (finger rinse) and in thepresence of a SalmoKCaR agonist (gadolinium) and a repellant (fingerrinse). The figure shows that the olfactory nerve impulse to therepellant is reversibly altered in the presence of a SalmoKCaR agonist.

FIG. 44 shows the raw recordings from high resistance electrodes offreshwater adapted Atlantic Salmon in response to a series of repeatedstimuli (L-alanine or NaCl) in 2 minute intervals. The figure shows thatthe olfactory nerve impulse to the attractant is reversibly altered inthe presence of a SalmoKCaR agonist

FIG. 45 is a graphical representation of the ratio from FURA-2 cellsexpressing a PVCR in the presence or absence of 10 mM L-Isoleucine invarious concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 20.0 mM) ofextracellular calcium (Ca²⁺).

FIG. 46 is a graphical representation of the fractional Ca²⁺ response,as compared to the extracelluar Ca²⁺ (mM) for the PVCR in Ca²⁺ only,Phenylalanine, Isoleucine, or AA Mixture (a variety of L-isomers invarious concentrations).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to three novel isolated sequences fromPVCR genes, SalmoKCaR#1, SalmoKCaR#2, and SalmoKCaR#3, in AtlanticSalmon. These genes encode three polypeptide sequences that are also thesubject of the present invention. These polypeptide sequences allow foror assist in several functions including sensing at least one SalmoKCaRmodulator in serum or in the surrounding environment; adapting to atleast one SalmoKCaR modulator present in the serum or surroundingenvironment; imprinting Atlantic Salmon with an odorant; altering waterintake; altering water absorption; or altering urine output.

USES OF THE PRESENT INVENTION

One use of the present invention relates to methods for improving theraising of salmon and/or methods for preparing salmon for transfer fromfreshwater to seawater. These methods involve adding one or more PVCR(e.g., SalmoKCaR) modulators to the freshwater (e.g., calcium and/ormagnesium), and adding a specially made or modified feed to thefreshwater for consumption by the fish. The feed contains a sufficientamount of sodium chloride (NaCl) and/or a SalmoKCaR modulator (e.g., anamino acid like tryptophan) to significantly increase levels of theSalmoKCaR modulator in the serum. During this process, the serum levelof the SalmoKCaR modulator significantly increases in the salmon, andcauses modulated (e.g., increased and/or decreased) SalmoKCaR expressionand/or altered SalmoKCaR sensitivity. This process prepares salmon fortransfer to seawater, so that they can better adapt to seawater oncethey are transferred. The details of how to carry out this process isdescribed in the Exemplification Section. In particular, theExemplification describes two processes. Briefly, Process I involvesadding calcium and magnesium to the water, and providing feed containingNaCl; and Process II includes adding calcium and magnesium to the water,and providing feed having both NaCl and tryptophan. Studies performedand described in Example 7 show that Atlantic Salmon maintained infreshwater and subjected to Process I had a survival rate of 91%, andthose Atlantic Salmon subjected to Process II had a survival rate of99%; as compared to control fish having a survival rate of only 67%after transfer to seawater. Similarly, in the same experiment, five daysafter transfer to seawater, Atlantic Salmon subjected to Process I had asurvival rate of 90%, while Atlantic Salmon subjected to Process II hada survival rate of 99%. The control fish had a survival rate of only 50%after being transferred to seawater. Furthermore, experiments describedin Example 6 demonstrate that modulated expression of one or moreSalmoKCaR genes occurs in various tissues during Process I and ProcessII. Process I and II, as described herein, modulate the SalmoKCaR genesand allow for increased food consumption, growth and survival; anddecreased morbidity and susceptibility to disease.

Process I and II likely have further utility in restoration of wildAtlantic salmon populations. Since a major cause of mortality of wildAtlantic salmon smolt is loss or capture by predators as they areadapting to seawater in river estuaries, treatment of wild Atlanticsalmon produced in large numbers, as part of river restocking programswould boost the productivity and survival of fish produced in suchprograms. Moreover, several studies have shown that salmon smolt arealso poisoned by exposure to heavy metals (Al³⁺, Zn²⁺, Cu²⁺) thatcontaminate their native rivers in both the US and other countries suchas Norway. These highly deleterious effects on salmon are manifestedprincipally in rivers with low natural Ca²⁺ concentrations. Thus,treatment of wild strains of Atlantic salmon produced in restockinghatcheries with either Process I or Process II would render thesetreated smolt less susceptible to the effects of heavy metals since thesmoltification process in these treated smolt was much further advancedthat in untreated fish. Use of Process I or II to treat Atlantic salmonthat would be released into rivers also have commercial utility inlarge-scale ocean ranching programs where large numbers of salmon smoltare released and captured for human consumption upon their return from1-3 years in the ocean.

Similarly, since expression of the SalmoKCaR genes changes duringProcess I and Process II, assaying these genes allows one to determineif the salmon are ready for transfer to seawater. Examples of suchassays are ELISAs, radioimmunoassays (RIAs), southern blots and RT-PCRassays, which are described herein in detail. The salmon are subjectedto either Process I or Process II for a period of time in freshwaterbefore being transferred to seawater. The SalmoKCaR genes, orpolypeptides encoded by these genes, can be assayed for determining theoptimal time period for maintaining the salmon in the freshwater, beforetransfer to seawater. Using methods described herein, salmon can beassayed to determine if modulated levels of the SalmoKCaR genes and/orpolypeptides have occurred, as compared to controls. For example, whenfish that are maintained in freshwater and subjected to either Process Ior Process II and changes in one or more of SalmoKCaR genes and/orpolypeptide levels in at least one tissue are modulated such that theymimic changes in the same genes and/or polypeptide levels that would beseen in fish adapted to seawater, then this group of fish are ready tobe transferred to seawater. In one experiment, the increased expressionof SalmoKCaR genes in the kidney of Atlantic Salmon subjected to ProcessII was similar to the increased expression in the same tissue forAtlantic Salmon already adapted to seawater, but dissimilar toexpression to Atlantic Salmon adapted to freshwater (i.e., no increasedexpression in the kidney water fish was seen). See Example 6. Whenlevels of SalmoKCaR genes and/or polypeptide encoded by these genes aresimilar to those levels seen in fish that have been transferred toseawater, then in the experiments described herein, the transfer ofthese salmon result in several benefits including increased survival andgrowth. Also, the optimal time periods for subjecting salmon to ProcessI or Process II are generally between 4-6 weeks, but vary depending onthe strain of salmon or process used. Hence, the assays described hereincan be used to determine the optimal amount of time for subjecting thesalmon to either Process I or Process II before transferring toseawater.

Additionally, comparison of the SalmoKCaR #3 sequence with datagenerated from site directed mutagenesis studies of mammalian CaRsindicates that the SalmoKCaR #3 protein likely generates a dominantnegative effect on the other SalmoKCaR #1 and #2 proteins when they areexpressed together in the same cell. This dominant negative effect ofSalmoKCaR #3 occurs since it lacks that necessary carboxyl terminaldomain to propagate signals generated by the binding of PVCR agonists.Interactions between the fully functional SalmoKCaR #1 or #2 proteinsand SalmoKCaR #3 would cause a marked reduction in the sensitivity ofthe SalmoKCaR #1 or #2 proteins. In one experiment, it was found thatincreased expression of SalmoKCaR#3 was seen in tissues readily exposedto high concentrations of calcium and magnesium in the surroundingenvironment (e.g., gill and nasal lamellae) or tissues that excrete highconcentrations of calcium and magnesium (e.g., urinary bladder andkidney). Therefore, such assays can be used to determine levels of theindividual SalmoKCaR genes, and compare expression levels to oneanother, and to individual levels of these genes of seawater adaptedsalmon to determine whether the salmon being tested are ready fortransfer to seawater.

Uses of nucleic acids of the present invention include one or more ofthe following: (1) producing receptor proteins which can be used, forexample, for structure determination, to assay a molecule's activity,and to obtain antibodies binding to the receptor; (2) being sequenced todetermine a receptor's nucleotide sequence which can be used, forexample, as a basis for comparison with other receptors to determine oneor more of the following: conserved sequences; unique nucleotidesequences for normal and altered receptors; and nucleotide sequences tobe used as target sites for antisense nucleic acids, ribozymes, or PCRamplification primers; (3) as hybridization detection probes to detectthe presence of a native receptor and/or a related receptor in a sample,as further described herein to determine the presence or level ofSalmoKCaR in a sample for, e.g., assessing whether salmon are ready fortransfer to seawater; (4) as PCR primers to generate particular nucleicacid sequence sequences, for example, to generate sequences to be usedas hybridization detection probes; and (5) for determining and isolatingadditional aquatic PVCR homologs in other species.

Another use for nucleic acid sequences of SalmoKCaRs #1, #2 or #3 is asprobes for the screening of Atlantic salmon broodstock, eggs, sperm,embryos or larval and juvenile fish as part of breeding programs. Use ofSalmoKCaR probes would enable identification of desirable traits such asenhanced salinity responsiveness, homing, growth in seawater orfreshwater or improve the feed utilization that were due to orassociated with naturally occurring or induced mutations of SalmoKCaRgenes. Nucleic acid sequences of SalmoKCaRs #1, #2 or #3 can also beused as probes for screening of wild Atlantic salmon in various regionsas a tool to identify specific strains of fish from both sea run andland locked strains. Such strains could then be used to interbreed withexisting commercial strains to produce further improvements in fishperformance.

The structural-functional data generated via study of recombinantSalmoKCaRs after their expression in cells as functional proteins can beused to identify desirable alternations in the function of SalmoKCaRproteins that could then be screened for as part of geneticselection-broodstock enhancement program.

Cell lines expressing SalmoKCaR proteins, either individually or invarious combinations, would have utility and value as a means to assayvarious compounds, chemicals and water conditions that occur both in thenatural and commercial environments. Utilization of transfected cellsexpressing SalmoKCaR #1-3 proteins either alone or in variouscombinations can be used in screening methods to identify both naturallyoccurring and commercially synthesized compounds that would enhance theperformance of wild or commercially produced Atlantic salmon includingsalinity adaption, feeding, growth and maturation, flesh quality, homingto areas of spawning, recognition of specific odorants as part ofimprinting, utilization of nutrients with improved efficiency andaltered behavior. Such screening assay would be a vast improvement overexisting assays where large numbers of fish are required and their endresponse (e.g., behavior, feeding, growth, survival or appearance isaltered) to a given compound produce complicated assays that have manyproblems with data interpretation. Transfected cells expressingSalmoKCaR #1-3 proteins either alone or in various combinations can alsobe used in screening methods to screen for specific water conditionsincluding pH, ionic strength and composition of various compoundsdissolved in the water to alter the function of SalmoKCaR proteins andthus lead to improved salinity responses in various life stages ofAtlantic salmon. Such assays would be designed to determine theinteractions and effects of these conditions on SalmoKCaR proteinswithout having to test the effects of such compounds on either wholeliving fish or some tissue explants.

Fragments of recombinant SalmoKCaR proteins also provide a utility asmodulators of PVCR function that could be added to water, applied totissue surfaces such as gills or skin or injected into fish via standardtechniques. The present invention is also useful in immunization of anyone of the various life stages of Atlantic salmon (eggs, embryo, larvalor juvenile or adult fish with either whole or fragments of recombinantSalmoKCaR proteins to create antibody responses that would, in turn,alter SalmoKCaR mediated functions of fish.

The present invention is not limited to the uses described in thissection. Based on the data and information described herein, additionaluses of the present invention may be readily appreciated by one of skillin the art.

The SalmoKCaR Polypeptides and its Function

The present invention relates to isolated polypeptide molecules thathave been isolated in Atlantic Salmon including three full lengthsequences. The present invention includes polypeptide molecules thatcontain the sequence of any one of the full length SalmoKCaR amino acidsequence (SEQ ID NO: 8, 10, or 12). See FIGS. 9, 10 and 11. The presentinvention also pertains polypeptide molecules that are encoded bynucleic acid molecules having the sequence of any one of the isolatedfull length SalmoKCaR nucleic acid sequences (SEQ ID NO: 7, 9, or 11).

SalmoKCaR polypeptides referred to herein as “isolated” are polypeptidesthat separated away from other proteins and cellular material of theirsource of origin. Isolated SalmoKCaR proteins include essentially pureprotein, proteins produced by chemical synthesis, by combinations ofbiological and chemical synthesis and by recombinant methods. Theproteins of the present invention have been isolated and characterizedas to its physical characteristics using laboratory techniques common toprotein purification, for example, salting out, immunoprecipation,column chromatography, high pressure liquid chromatography orelectrophoresis. SalmoKCaR proteins are found in many tissues in fishincluding gill, nasal lamellae, urinary bladder, kidney, stomach,pyloric caeca, proximal intestine, distal intestine, brain, pituitarygland, olfactory bulb, liver, muscle, skin and brain.

The present invention also encompasses SalmoKCaR proteins andpolypeptides having amino acid sequences analogous to the amino acidsequences of SalmoKCaR polypeptides. Such polypeptides are definedherein as SalmoKCaR analogs (e.g., homologues), or mutants orderivatives. “Analogous” or “homolgous” amino acid sequences refer toamino acid sequences with sufficient identity of any one of theSalmoKCaR amino acid sequences so as to possess the biological activityof any one of the native SalmoKCaR polypeptides. For example, an analogpolypeptide can be produced with “silent” changes in the amino acidsequence wherein one, or more, amino acid residues differ from the aminoacid residues of any one of the SalmoKCaR protein, yet still possessesthe function or biological activity of the SalmoKCaR. Examples of suchdifferences include additions, deletions or substitutions of residues ofthe amino acid sequence of SalmoKCaR. Also encompassed by the presentinvention are analogous polypeptides that exhibit greater, or lesser,biological activity of any one of the SalmoKCaR proteins of the presentinvention. Such polypeptides can be made by mutating (e.g.,substituting, deleting or adding) one or more amino acid or nucleic acidresidues to any of the isolated SalmoKCaR molecules described herein.Such mutations can be performed using methods described herein and thoseknown in the art. In particular, the present invention relates tohomologous polypeptide molecules having at least about 70% (e.g., 75%,80%, 85%, 90% or 95%) identity or similarity with SEQ ID NO: 8, 10, or12. Percent “identity” refers to the amount of identical nucleotides oramino acids between two nucleotides or amino acid sequences,respectfully. As used herein, “percent similarity” refers to the amountof similar or conservative amino acids between two amino acid sequences.Each of the SalmoKCaR polypeptides are homologous to one another.

The percent identity when comparing one SalmoKCaR amino acid sequence toanother are as follows: Percent Identity for Amino Acid Sequences* QuerySequence SalmoKCaR#1 SalmoKCaR#2 SalmoKCaR#3 SalmoKCaR#1 N/A 99.9% 89.6%SalmoKCaR#2 99.9% N/A 89.5% SalmoKCaR#3 99.2% 99.1% N/A*Note that the percentages are based on the number of aa's in the targetsequence.

The polypeptides of the present invention, including the full lengthsequences, the partial sequences, functional fragments and homologues,allow for or assist in one or more of the following functions: sensingat least one SalmoKCaR modulator in serum or in the surroundingenvironment; adapting to at least one SalmoKCaR modulator present in theserum or surrounding environment; imprinting Atlantic Salmon with anodorant; altering water intake; altering water absorption; alteringurine output. These and additional functions of the polypeptides arefurther described herein, and illustrated by the Exemplification. Theterm “sense” or “sensing” refers to the SalmoKCaR's ability to alter itsexpression and/or sensitivity in response to a SalmoKCaR modulator.

Homologous polypeptides can be determined using methods known to thoseof skill in the art. Initial homology searches can be performed at NCBIagainst the GenBank, EMBL and SwissProt databases using, for example,the BLAST network service. Altschuler, S. F., et al., J. Mol. Biol.,215:403 (1990), Altschuler, S. F., Nucleic Acids Res., 25:3389-3402(1998). Computer analysis of nucleotide sequences can be performed usingthe MOTIFS and the FindPatterns subroutines of the Genetics ComputingGroup (GCG, version 8.0) software. Protein and/or nucleotide comparisonswere performed according to Higgins and Sharp (Higgins, D. G. and Sharp,P. M., Gene, 73:237-244 (1988) e.g., using default parameters).

The SalmoKCaR proteins of the present invention also encompassbiologically active or functional polypeptide fragments of the fulllength SalmoKCaR proteins. Such fragments can include the partialisolated amino acid sequences (SEQ ID NO: 15 and 27), or part of thefull-length amino acid sequence (SEQ ID NO: 8, 10, or 12), yet possessthe function or biological activity of the full length sequence. Forexample, polypeptide fragments comprising deletion mutants of theSalmoKCaR proteins can be designed and expressed by well-knownlaboratory methods. Fragments, homologues, or analogous polypeptides canbe evaluated for biological activity, as described herein.

In one embodiment, the function or biological activity relates topreparing salmon for transfer to seawater. The method for preparingAtlantic Salmon for transfer to seawater includes adding at least oneSalmoKCaR modulator (e.g., PVCR modulator) to the freshwater, and addinga specially made or modified feed to the freshwater for consumption bythe fish. The feed contains a sufficient amount of sodium chloride(NaCl) (e.g., between about 1% and about 10% by weight, or about 10,000mg/kg to about 100,000 mg/kg) to significantly increase levels of theSalmoKCaR modulator in the serum. This amount of NaCl in the feed causesor induces the Atlantic Salmon to drink more freshwater. Since thefreshwater contains a SalmoKCaR modulator and the salmon ingestincreased amounts of it, the serum level of the SalmoKCaR modulatorsignificantly increases in the salmon, and causes modulated (e.g.,increased and/or decreased) SalmoKCaR expression and/or alteredSalmoKCaR sensitivity. One function or activity of the SalmoKCaR genesis to sense SalmoKCaR modulators in the serum. The SalmoKCaR expressionis altered by the SalmoKCaR modulators in the serum, which provides theability for the salmon to better adapt to seawater, undergosmoltification, survive, grow, consume food and/or to be lesssusceptible to disease.

A “PVCR modulator” or “SalmoKCaR modulator” refers to a compound whichmodulates (e.g., increases and/or decreases) expression of SalmoKCaR, oralters the sensitivity or responsiveness of SalmoKCaR genes. Suchcompounds include, but are not limited to, SalmoKCaR agonists (e.g.,inorganic polycations, organic polycations and amino acids), Type IIcalcimimetics, and compounds that indirectly alter PVCR expression(e.g., 1,25 dihydroxyvitamin D in concentrations of about 3,000-10,000International Units/kg feed), cytokines such as Interleukin Beta, andMacrophage Chemotatic Peptide-1 (MCP-1)). Examples of Type IIcalcimimetics, which increase and/or decrease expression, and/orsensitivity of the SalmoKCaR genes, are, for example, NPS-R-467 andNPS-R-568 from NPS Pharmaceutical Inc., (Salt Lake, Utah, U.S. Pat. Nos.5,962,314; 5,763,569; 5,858,684; 5,981,599; 6,001,884) which can beadministered in concentrations of between about 0.1 μM and about 100 μMfeed or water. See Nemeth, E. F. et al., PNAS 95: 4040-4045 (1998).Examples of inorganic polycations are divalent cations including calciumat a concentration between about 2.0 and about 10.0 mM and magnesium ata concentration between about 0.5 and about 10.0 mM; and trivalentcations including, but not limited to, gadolinium (Gd3+) at aconcentration between about 1 and about 500 μM. Organic polycationsinclude, but are not limited to, aminoglycosides such as neomycin orgentamicin in concentrations of between about 1 and about 8 gm/kg feedas well as organic polycations including polyamines (e.g., polyarginine,polylysine, polyhistidine, polyornithine, spermine, spermidine,cadaverine, putrescine, copolymers of poly arginine/histidine, polylysine/arginine in concentrations of between about 10 μM and 10 mMfeed). See Brown, E. M. et al., Endocrinology 128: 3047-3054 (1991);Quinn, S. J. et al., Am. J. Physiol. 273: C1315-1323 (1997).Additionally, SalmoKCaR agonists include amino acids such asL-Tryptophan L-Tyrosine, L-Phenylalanine, L-Alanine, L-Serine,L-Arginine, L-Histidine, L-Leucine, L-Isoleucine, L-Aspartic acid,L-Glutamic acid, L-Glycine, L-Lysine, L-Methionine, L-Asparagine,L-Proline, L-Glutamine, L-Threonine, L-Valine, and L-Cysteine atconcentrations of between about 1 and about 10 gm/kg feed. SeeConigrave, A. D., et al., PNAS 97: 4814-4819 (2000). Amino acids, in oneembodiment, are also defined as those amino acids that can be sensed byat least one SalmoKCaR in the presence of low levels of extracellularcalcium (e.g., between about 1 mM and about 10 mM). In the presence ofextracellular calcium, the SalmoKCaR in organs or tissues such as theintestine, pyloric caeca, or kidney can better sense amino acids. Themolar concentrations refer to free or ionized concentrations of theSalmoKCaR modulator in the freshwater, and do not include amounts ofbound SalmoKCaR modulator (e.g., SalmoKCaR modulator bound to negativelycharged particles including glass, proteins, or plastic surfaces). Anycombination of these modulators can be added to the water or to the feed(in addition to the NaCl, as described herein), so long as thecombination modulates expression and/or sensitivity of one or more ofthe SalmoKCaR genes.

Another function of the SalmoKCaR polypeptides involves imprintingAtlantic Salmon with an odorant (e.g., an attractant or repellant).Atlantic Salmon can be imprinted with an odorant so that, when the fishare later exposed to the odorant, they can more easily distinguish theodorant or are sensitized to the odorant. The SalmoKCaR polypeptides canwork, for example, with one or more olfactory receptors to modify thegeneration of the nerve impulse during sensing of an odorant. Generationof this nerve impulse occurs upon binding of the odorant to theolfactory lamellae in the fish. The SalmoKCaR modulator alters theolfactory sensing of the salmon to the odorant. In some cases, thepresence of a (e.g., at least one) SalmoKCaR modulator in freshwaterreversibly reduces or ablates the fish's ability to sense certainodorants. In other cases it can be heightened or increased. By exposingthe salmon in freshwater having a SalmoKCaR modulator to an odorant, thefish have an altered response which depending on the modulator wouldconsist of either a decreased or heightened response to the odorant.Briefly, these imprinting methods involve adding at least one SalmoKCaRmodulator (e.g., calcium and magnesium) to the freshwater in an amountsufficient to modulate expression and/or sensitivity of at least oneSalmoKCaR gene; and adding feed for fish consumption to the freshwater.The feed contains at least one an attractant (e.g., alanine); an amountof NaCl sufficient to contribute to a significantly increased level ofthe SalmoKCaR modulator in serum of the Atlantic Salmon; and optionallya SalmoKCaR modulator (e.g., tryptophan). The odorant can also be addedto the water, instead of the feed. Salmon that has been imprinted withan attractant consume more feed having this attractant and, as a result,grow faster. The imprinting process occurs during various developmentalstages of salmon including the larval stage and the smoltificationstage. Localizations of SalmoKCaR proteins and detection of SalmoKCaRexpression using RT-PCR in various organs involved in the imprintingprocess including olfactory lamellae, olfactory bulb and brain isprovided for both larval (Example 13) and smolt stages (FIGS. 34 and35). The process of imprinting the salmon with an odorant refers tocreating a lasting effect or impression on the fish so that the fish aresensitized to the odorant or can distinguish the odorant. Beingsensitized to the odorant refers to the fish's ability to more easilyrecognize or recall the odorant. Distinguishing an odorant refers to thefish's ability to differentiate among one or more odorants, or have apreference for one odorant over another.

An odorant is a compound that binds to olfactory receptors and causesfish to sense odorants. Generation of an olfactory nerve impulse occursupon binding of the odorant to the olfactory lamellae. A fish odorant iseither a fish attractant or fish repellant. A fish attractant is acompound to which fish are attracted. The sensitivity of the attractantis modulated, at least in part, by the sensitivity and/or expression ofthe SalmoKCaR genes in the olfactory apparatus of the fish in responseto a SalmoKCaR modulator. Examples of attractants in some fish includeamino acids (e.g., L-Tryptophan L-Tyrosine, L-Phenylalanine, L-Alanine,L-Serine, L-Arginine, L-Histidine, L-Leucine, L-Isoleucine, L-Asparticacid, L-Glutamic acid, L-Glycine, L-Lysine, L-Methionine, L-Asparagine,L-Proline, L-Glutamine, L-Threonine, L-Valine, and L-Cysteine),nucleotides (e.g., inosine monophosphate), organic compounds (e.g.,glycine-betaine and trimethylamine oxide), or a combination thereof.Similarly, a fish repellant is a compound that fish are repelled by, andthe sensitivity of the fish to the repellant is altered throughexpression and/or sensitivity of a SalmoKCaR gene in the olfactoryapparatus of the fish in the presence of a SalmoKCaR modulator. Anexample of a repellant is a “finger rinse” which is a mixture ofmammalian oils and fatty acids produced by the epidermal cells of theskin, and is left behind after human fingers are rinsed with an aqueoussolution. Methods for performing a finger rinse is known in the art andis described in more detailed in the Exemplification Section.

Additionally, the function of SalmoKCaR polypeptides includes itsability to sense or adapt to ion concentrations in the surroundingenvironment. The SalmoKCaR polypeptides sense various SalmoKCaRmodulators including calcium, magnesium and/or sodium. The SalmoKCaRpolypeptides are modulated by varying ion concentrations. For instance,any one of the SalmoKCaR polypeptides can be modulated (e.g., increasedor decreased) in response to a change in ion concentration (e.g.,calcium, magnesium, or sodium). Responses to changes in ionconcentrations of Atlantic Salmon containing the SalmoKCaR polypeptidesinclude the ability to adapt to the changing ion concentration. Suchresponses include the amount the fish drinks, the amount of urineoutput, and the amount of water absorption. Responses also includechanges in biological processes that affect its ability to excretecontaminants.

More specifically, methods are available to regulate salinity tolerancein fish by modulating (e.g., increasing, decreasing or maintaining theexpression) the activity of one or more of the SalmoKCaR proteinspresent in cells involved in ion transport. For example, salinitytolerance of fish adapted (or acclimated) to freshwater can be increasedby activating one or more of the SalmoKCaR polypeptides, for example, byincreasing the expression of one or more of SalmoKCaR genes, resultingin the secretion of ions and seawater adaption. Alternatively, thesalinity tolerance of fish adapted to seawater can be decreased byinhibiting one or more of the SalmoKCaR proteins, resulting inalterations in the absorption of ions and freshwater adaption.

“Salinity” refers to the concentration of various ions in a surroundingaquatic environment. In particular, salinity refers to the ionicconcentration of calcium, magnesium and/or sodium (e.g., sodiumchloride). “Normal salinity” levels refers to the range of ionicconcentrations of typical water environment in which an aquatic speciesnaturally lives. Normal salinity or normal seawater concentrations areabout 10 mM Ca, about 40 mM Mg, and about 450 mM NaCl. “Salinitytolerance” refers to the ability of a fish to live or survive in asalinity environment that is different than the salinity of its naturalenvironment. Modulations of the PVCR allows fish to live in about fourtimes and one-fiftieth, preferably, twice and one-tenth the normalsalinity.

The ability of anadromous fish (Atlantic salmon, trout and Arctic char)as well as euryhaline fish (flounders, alewives, eels) to traverse fromfreshwater to seawater environments and back again is of key importanceto their lifecycles in the natural environment. Both types of fish haveto undergo similar physiological changes including alterations in theirurine output, altering water intake and water absorption. Both types offish utilize environments of either freshwater (Atlantic salmon) orpartial salinity (flounders) to spawn and allow for the development oflarval fish into juvenile forms that then undergo changes to migrateinto full strength seawater. Both types of fish utilize PVCRs to sensewhen adult fish have arrived in a salinity environment suitable forspawning and to guide their return back to full strength seawater.Similarly, their resulting offspring utilize PVCRs to control variousorgans allowing for their normal development in fresh or brackish(partial strength seawater) water and subsequently to regulate thephysiological changes that permit these fish to migrate into fullstrength seawater.

The following experiment was done in Summer and Winter Flounder, but isapplicable to Atlantic Salmon because both species of fish have PVCRswhich respond to ion concentrations in a similar manner. Summer andWinter Flounder were adapted to live in 1/10th seawater (100 mOsm/kg) byreduction in salinity from 450 mM NaCl to 45 mM NaCl over an interval of8 hrs. Summer and Winter Flounder can be maintained in 1/10 or twice thesalinity for over a period of 6 months. After a 10 day interval wherethe Summer and Winter Flounder were fed a normal diet, the distributionof the PVCR in their urinary bladder epithelial cells was examined usingimmunocytochemistry. PVCR immunostaining is reduced and localizedprimarily to the apical membrane of epithelial cells in the urinarybladder. In contrast, the distribution of PVCR in epithelial cellslining the urinary bladders of control flounders continuously exposed tofull strength seawater is more abundant and present in both the apicalmembranes as well as in punctate sequences throughout the cell. Thesedata are consistent with previous Northern data where more PVCR proteinis present in the urinary bladders of seawater fish vs fish adapted tobrackish water. These data show that PVCR protein is expressed inepithelial cells that line the urinary bladder where the PVCR proteincomes into direct contact with the urine that is being formed by thekidney. Due to its location in the cell membrane of these epithelialcells, the PVCR proteins can “sense” changes in the urine's compositionon a continuous basis. Depending on the specific ionic concentrations ofthe urine, the PVCR protein alters the transport of ions across theepithelium of the urinary bladder and, in this way, determines the finalcomposition of the urine. This composition and the amount of water andNaCl absorbed from the urine are critical to salinity regulation infish.

As urinary magnesium and calcium concentrations increase when fish arepresent in full strength sea water, activation of apical PVCR proteincauses reduction in urinary bladder water transport. The inventionprovides methods to facilitate euryhaline adaptation of fish to occur,and improve the adaption. More specifically, methods are now availableto regulate salinity tolerance in fish by modulating (e.g., alternating,activating and or expressing) the activity of the PVCR protein presentin epithelial cells involved in ion transport, as well as in endocrineand nervous tissue. For example, salinity tolerance of fish adapted (oracclimated) to fresh water can be increased by activating the PVCR, forexample, by increasing the expression of PVCR in selected epithelialcells, resulting in the secretion of ions and seawater adaption.Specifically, this would involve regulatory events controlling theconversion of epithelial cells of the gill, intestine and kidney. In thekidney, PVCR activation facilitates excretion of divalent metal ionsincluding calcium and magnesium by renal tubules. In the gill, PVCRactivation reduces reabsorption of ions by gill cells that occurs infresh water and promote the net excretion of ions by gill epithelia thatoccurs in salt water. In the intestine, PVCR activation will permitreabsorption of water and ions across the G.I. tract after theiringestion by fish.

Alternatively, the salinity tolerance of fish adapted to seawater can bedeceased by modulating one or more of the SalmoKCaR polypeptides, forexample by decreasing the expression of one or more of the SalmoKCaRgenes while others may be increased. The net result of these changeswould be alterations in the absorption of ions that facilitate theadaption to freshwater conditions.

In another example, Winter and Summer Flounder were maintained in atleast twice the normal salinity or 1/10 the normal salinity. SeeExemplification. These fish can be maintained in these environments forlong periods of time (e.g., over 3 months, over 6 months, or over 1year). These limits were defined by decreasing or increasing the ionicconcentrations of calcium, magnesium, and sodium, keeping a constantratio between the ions. These salinity limits can be further defined byincreasing and/or decreasing an individual ion concentration, therebychanging the ionic concentration ratio among the ions. Increasing and/ordecreasing individual ion concentrations can increase and/or decreasesalinity tolerance. “Hypersalinity” or “above normal salinity” levelsrefers to a level of at least one ion concentration that is above thelevel found in normal salinity. “Hyposalinity” or “below normalsalinity” levels refers to a level of at least one ion concentrationthat is below the level found in normal salinity.

Maintaining fish in a hypersalinity environment also results in fishwith a reduced number of parasites or bacteria. Preferably, theparasites and/or bacteria are reduced to a level that is safe for humanconsumption, raw or cooked. More preferably, the parasites and/orbacteria are reduced to having essentially no parasites and fewbacteria. These fish must be maintained in a hypersalinity environmentlong enough to rid the fish of these parasites or bacteria, (e.g., forat least a few days or at least a few weeks).

The host range of many parasites is limited by exposure to watersalinity. For example, Diphyllobothrium species commonly known as fishtapeworms, is encountered in the flesh of fish, primarily fresh water orcertain euryhaline species. Foodborne Pathogenic Microorganisms andNatural Toxins Handbook. 1991. US Food and Drug Administration Centerfor Food Safety and Applied Nutrition, the teachings of which areincorporated herein by reference in their entirety. In contrast, itspresence in the flesh of completely marine species is much reduced orabsent. Since summer flounder can survive and thrive at salinityextremes as high as 58 ppt (1.8 times normal seawater) for extendedperiods in recycling water, exposure of summer flounder to hypersalinityconditions might be used as a “biological” remediation process to ensurethat no Diphyllobothrium species are present in the GI tract of summerflounder prior to their sale as product.

Data from Cole et al., (J. Biol. Chem. 272:12008-12013 (1997)), showthat winter flounder elaborate an antimicrobial peptide from their skinto prevent bacterial infections. Their data reveals that in the absenceof pleurocidin, Escherichia coli are killed by high concentrations ofNaCl. In contrast, low concentrations of NaCl (<300 mM NaCl) allow E.coli to grow and under these conditions pleurocidin presumably helps tokill them. These data provide evidence of NaCl killing of E. coli, aswell as highlight possible utility of bacterial elimination in fish.

Similarly, maintaining fish in a hyposalinity environment results in afish with a reduced amount of contaminants (e.g., hydrocarbons, aminesor antibiotics). Preferably, the contaminants are reduced to a levelthat is safe for human consumption, raw or cooked fish. More preferable,the contaminants are reduced to having essentially very littlecontaminants left in the fish. These fish must be maintained in ahyposalinity environment long enough to rid the fish of thesecontaminants, (e.g., for at least a few days or a few weeks).

Organic amines, such as trimethylamine oxide (TMAO) produce a “fishy”taste in seafood. They are excreted via the kidney in flounder. (Krogh,A., Osmotic Regulation in Aquatic Animals, Cambridge University Press,Cambridge, U.K. pgs 1-233 (1939), the teachings of which areincorporated herein by reference in their entirety). TMAO is synthesizedby marine organisms consumed by fish that accumulate the TMAO in theirtissues. Depending on the species of fish, the muscle content of TMAOand organic amines is either large accounting for the “strong” taste ofbluefish and herring or small such as in milder tasting flounder.

The presence of SalmoKCaR in brain reflects both its involvement inbasic neurotransmitter release via synaptic vesicles (Brown, E. M. etal., New England J. of Med., 333:234-240 (1995)), as well as itsactivity to trigger various hormonal and behavioral changes that arenecessary for adaptation to either fresh water or marine environments.For example, increases in water ingestion by fish upon exposure to saltwater is mediated by SalmoKCaR activation in a manner similar to thatdescribed for humans where PVCR activation by hypercalcemia in thesubfomical organ of the brain cause an increase in water drinkingbehavior (Brown, E. M. et al., New England J. of Med., 333:234-240(1995)). In fish, processes involving both alterations in serum hormonallevels and behavioral changes are mediated by the brain. These includethe reproductive and spawning activities of euryhaline fish in freshwater after their migration from salt water as well as detection ofsalinity of their environment for purposes of feeding, nesting,migration and spawning. The key events for successful reproduction inAtlantic salmon are to migrate to a specific streambed for spawningafter 1-3 years of free-swimming existence on the open ocean. Successfulachievement of this challenge depends on the combination of adult salmonbeing able to remember and navigate their way back to this originallocation as well as successful imprinting of larval and juvenileAtlantic salmon to odors present in freshwater in the freshwaterstreambed as well as the characteristics of the mouth of the river asthe fish exit the river and enter the ocean. Sensing of salinity by PVCRand its modulation of the odorant detection system of salmon fordetecting various odorants is critical to the achievement of theseprocesses.

Data obtained recently from mammals now suggest that PVCR activationplays a pivotal role in coordinating these events. For example,alterations in plasma cortisol have been demonstrated to be critical forchanges in ion transport necessary for adaptation of salmon smolts fromfresh water to salt water (Veillette, P. A., et al., Gen. and Comp.Physiol., 97:250-258 (1995)). As demonstrated recently in humans, plasmaAdrenocorticotrophic Hormone (ACTH) levels that regulate plasma cortisollevels are altered by PVCR activation.

Additionally, the function or biological activity of the SalmoKCaRpolypeptide or protein is defined, in one aspect, to mean theosmoregulatory activity of SalmoKCaR protein. Assay techniques toevaluate the biological activity of SalmoKCaR proteins and their analogsare described in Brown, et al., New Eng. J. Med., 333:243 (1995);Riccardi, et al., Proc. Nat. Acad. Sci USA, 92:131-135 (1995); andSands, et al., J. Clinical Investigation 99:1399-1405 (1997). Thebiological activity also includes the ability of the SalmoKCaR tomodulate signal transduction pathways in specific cells. Thus, dependingon the distribution and nature of various signal transduction pathwayproteins that are expressed in cells, biologically active SalmoKCaRproteins can modulate cellular functions in either an inhibitory orstimulatory manner.

Biologically active derivatives or analogs of the above describedSalmoKCaR polypeptides, referred to herein as peptide mimetics can bedesigned and produced by techniques known to those of skill in the art.(see e.g., U.S. Pat. Nos. 4,612,132; 5,643,873 and 5,654,276). Thesemimetics can be based, for example, on a specific SalmoKCaR amino acidsequence and maintain the relative position in space of thecorresponding amino acid sequence. These peptide mimetics possessbiological activity similar to the biological activity of thecorresponding peptide compound, but possess a “biological advantage”over the corresponding SalmoKCaR amino acid sequence with respect toone, or more, of the following properties: solubility, stability andsusceptibility to hydrolysis and proteolysis.

Methods for preparing peptide mimetics include modifying the N-terminalamino group, the C-terminal carboxyl group, and/or changing one or moreof the amino linkages in the peptide to a non-amino linkage. Two or moresuch modifications can be coupled in one peptide mimetic molecule.Modifications of peptides to produce peptide mimetics are described inU.S. Pat. Nos. 5,643,873 and 5,654,276. Other forms of the SalmoKCaRpolypeptides, encompassed by the present invention, include those whichare “functionally equivalent.” This term, as used herein, refers to anynucleic acid sequence and its encoded amino acid, which mimics thebiological activity of the SalmoKCaR polypeptides and/or functionaldomains thereof.

SalmoKCaR Nucleic Acid Sequences, Plasmids Vectors and Host Cells

The present invention, in one embodiment, includes an isolated fulllength nucleic acid molecule having a sequence of SalmoKCaR#1 (SEQ IDNO: 7), SalmoKCaR#2 (SEQ ID NO: 9) or SalmoKCaR#3 (SEQ ID NO: 11). SeeFIGS. 9, 10, and 11. The present invention includes sequences to thefull length SalmoKCaR nucleic acid sequences, as well as the codingregions thereof. As shown in these figures, the ORF SalmoKCaR#1 beginsat nt 180 and ends at nt 3005. For SalmoKCaR#2, it begins at nt 270 andends at nt 3095, and for SalmoKCaR#3, the ORF begins at nt 181 and endsat nt 2733.

The present invention also encompasses isolated nucleic acid sequencesthat encode SalmoKCaR polypeptides, and in particular, those whichencode a polypeptide molecule having an amino acid sequence of SEQ IDNO: 8, 10, or 12. The SalmoKCaR full length nucleic acid sequencesencode polypeptides that allow or assist in one or more of the followingfunctions: sensing at least one SalmoKCaR modulator in serum or in thesurrounding environment; adapting to at least one SalmoKCaR modulatorpresent in the serum or surrounding environment; imprinting AtlanticSalmon with an odorant; altering water intake; altering waterabsorption; or altering urine output.

The present invention encompasses the SalmoKCaR full length nucleic acidsequences, SalmoKCaR#1 (SEQ ID NO: 7), SalmoKCaR#2 (SEQ ID NO: 9), andSalmoKCaR#3 (SEQ ID NO: 11), or polypeptides encoded by these sequences,which were deposited under the Budapest Treaty with the ATCC, 10801University Boulevard, Manassas, Va. 20110-2209, USA on Mar. 29, 2002,under Accession Numbers PTA-4190, PTA-4191, and PTA-4192, respectively.These clones are plasmid DNA which can be transformed into E. Coli andcultured. The viability of the clones can be tested with ampicillinresistance. The sequences of the present invention can be purified fromthese deposits using techniques known in the art.

As used herein, an “isolated” gene or nucleotide sequence which is notflanked by nucleotide sequences which normally (e.g., in nature) flankthe gene or nucleotide sequence (e.g., as in genomic sequences) and/orhas been completely or partially purified from other transcribedsequences (e.g., as in a cDNA or RNA library). Thus, an isolated gene ornucleotide sequence can include a gene or nucleotide sequence which issynthesized chemically or by recombinant means. Nucleic acid constructscontained in a vector are included in the definition of “isolated” asused herein. Also, isolated nucleotide sequences include recombinantnucleic acid molecules and heterologous host cells, as well as partiallyor substantially or purified nucleic acid molecules in solution. In vivoand in vitro RNA transcripts of the present invention are alsoencompassed by “isolated” nucleotide sequences. Such isolated nucleotidesequences are useful for the manufacture of the encoded SalmoKCaRpolypeptide, as probes for isolating homologues sequences (e.g., fromother mammalian species or other organisms), for gene mapping (e.g., byin situ hybridization), or for detecting the presence (e.g., by Southernblot analysis) or expression (e.g., by Northern blot analysis) ofrelated genes in cells or tissue.

The SalmoKCaR nucleic acid sequences of the present invention includehomologues nucleic acid sequences. “Analogous” or “homologous” nucleicacid sequences refer to nucleic acid sequences with sufficient identityof any one of the SalmoKCaR nucleic acid sequences, such that onceencoded into polypeptides, they possess the biological activity of anyone of the native SalmoKCaR polypeptides. For example, an analogousnucleic acid molecule can be produced with “silent” changes in thesequence wherein one, or more, nt differ from the nt of any one of theSalmoKCaR protein, yet, once encoded into a polypeptide, still possessesthe function or biological activity of any one of the native SalmoKCaR.Examples of such differences include additions, deletions orsubstitutions. Also encompassed by the present invention are nucleicacid sequences that encode analogous polypeptides that exhibit greater,or lesser, biological activity of the SalmoKCaR proteins of the presentinvention. In particular, the present invention is directed to nucleicacid molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or95%) identity with SEQ ID NO: 8, 10, or 12. Each of the SalmoKCaR genesare homologues to one another.

The percent identity for the SalmoKCaR nucleic acid sequences are asfollows: Percent Identity for Nucleic Acid Sequences Query SequenceSalmoKCaR#1 SalmoKCaR#2 SalmoKCaR#3 SalmoKCaR#1 N/A 99.8% 95.8%SalmoKCaR#2 97.6% N/A 93.6% SalmoKCaR#3 98.7% 98.7% N/A

The nucleic acid molecules of the present invention, including the fulllength sequences, the partial sequences, functional fragments andhomologues, once encoded into polypeptides, allow for or assist in oneor more of the following functions: sensing at least one SalmoKCaRmodulator in serum or in the surrounding environment; adapting to atleast one SalmoKCaR modulator present in the serum or surroundingenvironment; imprinting Atlantic Salmon with an odorant; altering waterintake; altering water absorption; or altering urine output. Thehomologous nucleic acid sequences can be determined using methods knownto those of skill in the art, and by methods described herein includingthose described for determining homologous polypeptide sequences.

Also encompassed by the present invention are nucleic acid sequences,DNA or RNA, which are substantially complementary to the DNA sequencesencoding the SalmoKCaR polypeptides and which specifically hybridizewith their DNA sequences under conditions of stringency known to thoseof skill in the art. As defined herein, substantially complementarymeans that the nucleic acid need not reflect the exact sequence of theSalmoKCaR sequences, but must be sufficiently similar in sequence topermit hybridization with SalmoKCaR nucleic acid sequence under highstringency conditions. For example, non-complementary bases can beinterspersed in a nucleotide sequence, or the sequences can be longer orshorter than the SalmoKCaR nucleic acid sequence, provided that thesequence has a sufficient number of bases complementary to the SalmoKCaRsequence to allow hybridization therewith. Conditions for stringency aredescribed in e.g., Ausubel, F. M., et al., Current Protocols inMolecular Biology, (Current Protocol, 1994), and Brown, et al., Nature,366:575 (1993); and further defined in conjunction with certain assays.

The SalmoKCaR sequence, or a fragment thereof, can be used as a probe toisolate additional homologues. Nucleic acids encoding SalmoKCaRpolypeptides were identified by screening a cDNA library with aSalmoKCaR-specific probe under conditions known to those of skill in theart to identify homologous receptor proteins. For example, the fulllength sequences were isolated by screening Atlantic Salmon intestinaland kidney cDNA libraries with a probe consisting of a 653 nt PCRamplified genomic sequence (SEQ ID NO: 3). Techniques for thepreparation and screening of a cDNA library are well-known to those ofskill in the art. For example, techniques such as those described inRiccardi, et al., Proc. Nat. Acad. Sci. USA, 92:131-135 (1995), can beused. Positive clones can be isolated, subcloned and their sequencesdetermined. Using the sequences of either a full length or severalover-lapping partial cDNAs, the complete nucleotide sequence of theSalmoKCaR cDNA were obtained and the encoded amino acid sequencededuced. The sequences of the SalmoKCaRs can be compared to each otherand other aquatic PVCRs to determine differences and similarities.Methods for screening and identifying homologues genes as described ine.g., Ausubel, F. M., et al., Current Protocols in Molecular Biology,(Current Protocol, 1994).

SalmoKCaR genes were isolated by Polymerase Chain Reaction (PCR) ofgenomic DNA with degenerate primers (SEQ ID NOS: 13 and 14) specific toa highly conserved sequence of calcium receptors that does not containintrons. For example, partial Atlantic Salmon clones were obtained byusing degenerate primers that permit selective amplification of asequence (nucleotides 2279-2934 of SKCaR) that is highly conserved inboth mammalian and shark kidney calcium receptors. See Exemplification.The degenerate primers (SEQ ID NOS: 13 and 14) amplify a sequence of 653base pairs that is present in the extracellular domain of calciumreceptors. This 653 nt sequence refers to SEQ ID NO: 3 with the additionof the sequence of the primers. The resulting amplified 653 bp fragmentwas ligated into a cloning vector and transformed into bacterial cellsfor growth, purification and sequencing. Additionally, SalmoKCaR genescan be isolated by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) after isolation of poly A+ RNA from aquatic species with thesame or similar degenerate primers. Methods of PCR and RT-PCR are wellcharacterized in the art (See generally, PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila, etal., Nucleic Acids Res., 19:4967 (1991); Eckert, et al., PCR Methods andApplications, 1:17 (1991); PCR (eds. McPherson, et al., IRL Press,Oxford); and U.S. Pat. No. 4,683,202. Poly A+ RNA can be isolated fromany tissue which contains one or more of SalmoKCaR polypeptides bystandard methods as described. Preferred tissue for polyA+ RNA isolationcan be determined using an antibody which is specific for the highlyconserved sequence of calcium receptors, by standard methods. Thepartial genomic or cDNA sequences derived from a SalmoKCaR gene areunique and, thus, can be used as a unique probe to isolate thefull-length cDNA from other species. Moreover, in one embodiment, thisDNA fragment serves as a basis for specific assay kits for detection ofSalmoKCaR expression in various tissues of Atlantic Salmon.

Also encompassed by the present invention are nucleic acid sequences,genomic DNA, cDNA, RNA or a combination thereof, which are substantiallycomplementary to the DNA sequences encoding SalmoKCaR nucleic acidmolecules and which specifically hybridize with the SalmoKCaR nucleicacid sequences under conditions of sufficient stringency (e.g., highstringency) to identify DNA sequences with substantial nucleic acididentity.

The present invention embodies nucleic acid molecules (e.g., probes orprimers) that hybridize to SEQ ID NO: 7, 9, or 11 under high stringencyconditions, as defined herein. In one aspect, the present inventionincludes molecules that hybridize to at least about 200 contiguousnucleotides or longer in length (e.g., 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200,3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000). Such moleculeshybridize to one of the SalmoKCaR nucleic acid sequences (SEQ ID NO: 7,9, or 11) under high stringency conditions. The present inventionincludes those molecules that hybridize with SalmoKCaR nucleic acidmolecules and encode a polypeptide that has the functions or biologicalactivity described herein.

Typically the nucleic acid probe comprises a nucleic acid sequence (e.g.SEQ ID NO: 7, 9, or 11) and is of sufficient length and complementarityto specifically hybridize to a nucleic acid sequence that encodes aSalmoKCaR polypeptide. For example, a nucleic acid probe can be at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% the length ofthe SalmoKCaR nucleic acid sequence. The requirements of sufficientlength and complementarity can be easily determined by one of skill inthe art. Suitable hybridization conditions (e.g., high stringencyconditions) are also described herein. Additionally, the presentinvention encompasses fragments that are biologically active SalmoKCaRpolypeptides or nucleic acid sequences that encodes biologically activeSalmoKCaR polypeptides, as described further herein.

Such fragments are useful as probes for assays described herein, and asexperimental tools, or in the case of nucleic acid fragments, asprimers. A preferred embodiment includes primers and probes whichselectively hybridize to the nucleic acid constructs encoding any one ofthe SalmoKCaR proteins. For example, nucleic acid fragments which encodeany one of the domains described herein are also implicated by thepresent invention.

Stringency conditions for hybridization refers to conditions oftemperature and buffer composition which permit hybridization of a firstnucleic acid sequence to a second nucleic acid sequence, wherein theconditions determine the degree of identity between those sequenceswhich hybridize to each other. Therefore, “high stringency conditions”are those conditions wherein only nucleic acid sequences which are verysimilar to each other will hybridize. The sequences can be less similarto each other if they hybridize under moderate stringency conditions.Still less similarity is needed for two sequences to hybridize under lowstringency conditions. By varying the hybridization conditions from astringency level at which no hybridization occurs, to a level at whichhybridization is first observed, conditions can be determined at which agiven sequence will hybridize to those sequences that are most similarto it. The precise conditions determining the stringency of a particularhybridization include not only the ionic strength, temperature, and theconcentration of destabilizing agents such as formamide, but alsofactors such as the length of the nucleic acid sequences, their basecomposition, the percent of mismatched base pairs between the twosequences, and the frequency of occurrence of subsets of the sequences(e.g., small stretches of repeats) within other non-identical sequences.Washing is the step in which conditions are set so as to determine aminimum level of similarity between the sequences hybridizing with eachother. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between two sequences results in a1° C. decrease in the melting temperature (T_(m)) for any chosen SSCconcentration. Generally, a doubling of the concentration of SSC resultsin an increase in the T_(m) of about 17° C. Using these guidelines, thewashing temperature can be determined empirically, depending on thelevel of mismatch sought. Hybridization and wash conditions areexplained in Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) onpages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.

High stringency conditions can employ hybridization at either (1) 1×SSC(10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calfthymus DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumin(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with high stringency washes of either (1) 0.3-0.1×SSC, 0.1% SDSat 65° C., or (2) 1 mM Na₂EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS at 65° C.The above conditions are intended to be used for DNA-DNA hybrids of 50base pairs or longer. Where the hybrid is believed to be less than 18base pairs in length, the hybridization and wash temperatures should be5-10° C. below that of the calculated T_(m) of the hybrid, where T_(m)in ° C.=(2× the number of A and T bases)+(4× the number of G and Cbases). For hybrids believed to be about 18 to about 49 base pairs inlength, the T_(m) in ° C.=(81.5° C. +16.6(log₁₀M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

Moderate stringency conditions can employ hybridization at either (1)4×SSC, (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (2) 4×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumin(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with moderate stringency washes of 1×SSC, 0.1% SDS at 65° C. Theabove conditions are intended to be used for DNA-DNA hybrids of 50 basepairs or longer. Where the hybrid is believed to be less than 18 basepairs in length, the hybridization and wash temperatures should be 5-10°C. below that of the calculated T_(m) of the hybrid, where T_(m) in °C.=(2× the number of A and T bases)+(4× the number of G and C bases).For hybrids believed to be about 18 to about 49 base pairs in length,the T_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

Low stringency conditions can employ hybridization at either (1) 4×SSC,(10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calfthymus DNA at 50° C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 40° C., (3) 1% bovine serum albumin(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 50° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 40° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 50° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at40° C., with low stringency washes of either 2×SSC, 0.1% SDS at 50° C.,or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na₂EDTA, 40 mMNaHPO₄ (pH 7.2), 5% SDS. The above conditions are intended to be usedfor DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid isbelieved to be less than 18 base pairs in length, the hybridization andwash temperatures should be 5-10° C. below that of the calculated T_(m)of the hybrid, where T_(m) in ° C.=(2× the number of A and T bases)+(4×the number of G and C bases). For hybrids believed to be about 18 toabout 49 base pairs in length, the T_(m) in ° C.=(81.5°C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (% formamide)−500/L), where “M” is themolarity of monovalent cations (e.g., Na⁺), and “L” is the length of thehybrid in base pairs.

The SalmoKCaR nucleic acid sequence, or a fragment thereof, can also beused to isolate additional aquatic PVCR homologs. For example, a cDNA orgenomic DNA library from the appropriate organism can be screened withlabeled SalmoKCaR nucleic acid sequence to identify homologous genes asdescribed in e.g., Ausebel, et al., Eds., Current Protocols In MolecularBiology, John Wiley & Sons, New York (1997).

In another embodiment, the present invention pertains to a method ofisolating a SalmoKCaR nucleic acid comprising contacting an isolatednucleic acid with a SalmoKCaR-specific hybridization probe andidentifying an aquatic PVCR. Methods for identifying a nucleic acid byhybridization are routine in the art (see Current Protocols In MolecularBiology, Ausubel, F. M. et al., Eds., John Wiley & Sons: New York, N.Y.,(1997). The present method can optionally include a labeled SalmoKCaRprobe.

The invention also provides vectors, plasmids or viruses containing oneor more of the SalmoKCaR nucleic acid molecules. Suitable vectors foruse in eukaryotic and prokaryotic cells are known in the art and arecommercially available or readily prepared by a skilled artisan.Additional vectors can also be found, for example, in Ausubel, F. M., etal., Current Protocols in Molecular Biology, (Current Protocol, 1994)and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED.(1989).

Uses of plasmids, vectors or viruses containing the cloned SalmoKCaRreceptors or receptor fragments include one or more of the following;(1) generation of hybridization probes for detection and measuring levelof SalmoKCaR in tissue or isolation of SalmoKCaR homologs; (2)generation of SalmoKCaR mRNA or protein in vitro or in vivo; and (3)generation of transgenic non-human animals or recombinant host cells.

In one embodiment, the present invention encompasses host cellstransformed with the plasmids, vectors or viruses described above.Nucleic acid molecules can be inserted into a construct which can,optionally, replicate and/or integrate into a recombinant host cell, byknown methods. The host cell can be a eukaryote or prokaryote andincludes, for example, yeast (such as Pichia pastorius or Saccharomycescerevisiae), bacteria (such as E. coli or Bacillus subtilis), animalcells or tissue, insect Sf9 cells (such as baculoviruses infected SF9cells) or mammalian cells (somatic or embryonic cells, Human EmbryonicKidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human 293cells and monkey COS-7 cells). Host cells suitable in the presentinvention also include a fish cell, a mammalian cell, a bacterial cell,a yeast cell, an insect cell, and a plant cell.

The nucleic acid molecule can be incorporated or inserted into the hostcell by known methods. Examples of suitable methods of transfecting ortransforming cells include calcium phosphate precipitation,electroporation, microinjection, infection, lipofection and directuptake. “Transformation” or “transfection” as used herein refers to theacquisition of new or altered genetic features by incorporation ofadditional nucleic acids, e.g., DNA. “Expression” of the geneticinformation of a host cell is a term of art which refers to the directedtranscription of DNA to generate RNA which is translated into apolypeptide. Methods for preparing such recombinant host cells andincorporating nucleic acids are described in more detail in Sambrook etal., “Molecular Cloning: A Laboratory Manual,” Second Edition (1989) andAusubel, et al. “Current Protocols in Molecular Biology,” (1992), forexample.

The host cell is then maintained under suitable conditions forexpression and recovery of SalmoKCaR protein. Generally, the cells aremaintained in a suitable buffer and/or growth medium or nutrient sourcefor growth of the cells and expression of the gene product(s). Thegrowth media are not critical to the invention, are generally known inthe art and include sources of carbon, nitrogen and sulfur. Examplesinclude Luria broth, Superbroth, Dulbecco's Modified Eagles Media(DMEM), RPMI-1640, Ml 99 and Grace's insect media. The growth media cancontain a buffer, the selection of which is not critical to theinvention. The pH of the buffered Media can be selected and is generallyone tolerated by or optimal for growth for the host cell.

The host cell is maintained under a suitable temperature and atmosphere.Alternatively, the host cell is aerobic and the host cell is maintainedunder atmospheric conditions or other suitable conditions for growth.The temperature should also be selected so that the host cell toleratesthe process and can be for example, between about 13°-40° C.

Antibodies Fusion Proteins and Methods of Assessment of the SalmoKCaRNucleic Acid and Amino Acid Molecules

The present invention includes methods of detecting the levels of theSalmoKCaR nucleic acid levels (mRNA levels) and/or polypeptide levels todetermine whether fish are ready for transfer from freshwater toseawater. The present invention also includes methods for assayingcompounds that modulate SalmoKCaR nucleic acid levels, expression levelsor activity of SalmoKCaR polypeptides. Activity of SalmoKCaRpolypeptides includes, but is not limited to, phosphorylation of one ormore of the SalmoKCaR polypeptides, dimerization of one of the SalmoKCaRpolypeptides with a second SalmoKCaR polypeptide, proteolysis of one ormore of the SalmoKCaR polypeptides, and/or increase or decrease in theintracellular signal transduction system or pathway of one or more ofthe SalmoKCaR polypeptides. The present invention also includes assayingactivities, as known in the art. Methods that measure SalmoKCaR levelsinclude several suitable assays. Suitable assays encompass immunologicalmethods, such as FACS analysis, radioimmunoassay, flow cytometry,immunocytochemistry, enzyme-linked immunosorbent assays (ELISA) andchemiluminescence assays. Additionally, antibodies, or antibodyfragments, can also be used to detect the presence of SalmoKCaR proteinsand homologs in other tissues using standard immunohistological methods.For example, immunohistochemical studies were performed using the 1169antibody which was raised against a portion of the shark kidney calciumreceptor demonstrating localized expression in the olfactory organ.Antibodies are absorbed to determine the SalmoKCaR protein levels.Antibodies could be used in a kit to monitor the SalmoKCaR protein levelof fish in aquaculture. Any method known now or developed later can beused for measuring SalmoKCaR expression.

Antibodies reactive with any one of the SalmoKCaR or portions thereofcan be used. In a preferred embodiment, the antibodies specifically bindwith SalmoKCaR polypeptides or a portion thereof. The antibodies can bepolyclonal or monoclonal, and the term antibody is intended to encompasspolyclonal and monoclonal antibodies, and functional fragments thereof.The terms polyclonal and monoclonal refer to the degree of homogeneityof an antibody preparation, and are not intended to be limited toparticular methods of production.

In several of the preferred embodiments, immunological techniques detectSalmoKCaR levels by means of an anti-SalmoKCaR antibody (i.e., one ormore antibodies). The term “anti-SalmoKCaR” antibody includes monoclonaland/or polyclonal antibodies, and mixtures thereof.

Anti-SalmoKCaR antibodies can be raised against appropriate immunogens,such as isolated and/or recombinant SalmoKCaR, analogs or portionthereof (including synthetic molecules, such as synthetic peptides). Inone embodiment, antibodies are raised against an isolated and/orrecombinant SalmoKCaR or portion thereof (e.g., a peptide) or against ahost cell which expresses recombinant SalmoKCaR. In addition, cellsexpressing recombinant SalmoKCaR, such as transfected cells, can be usedas immunogens or in a screen for antibody which binds receptor.

Any suitable technique can prepare the immunizing antigen and producepolyclonal or monoclonal antibodies. The art contains a variety of thesemethods (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur.J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552(1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D.Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.); Current Protocols In MolecularBiology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al.,Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Generally, fusing a suitable immortal or myeloma cell line, such asSP2/0, with antibody producing cells can produce a hybridoma. Animalsimmunized with the antigen of interest provide the antibody producingcell, preferably cells from the spleen or lymph nodes. Selective cultureconditions isolate antibody producing hybridoma cells while limitingdilution techniques produce them. Researchers can use suitable assayssuch as ELISA to select antibody producing cells with the desiredspecificity.

Other suitable methods can produce or isolate antibodies of therequisite specificity. Examples of other methods include selectingrecombinant antibody from a library or relying upon immunization oftransgenic animals such as mice. Such methods include immunization ofvarious lifestages of Atlantic salmon to produce antibodies to nativePVCR proteins and thereby alter their function or specificity.

According to the method, an assay can determine the level of SalmoKCaRin a biological sample. In determining the amounts of SalmoKCaR, anassay includes combining the sample to be tested with an antibody havingspecificity for the SalmoKCaR, under conditions suitable for formationof a complex between antibody and the SalmoKCaR, and detecting ormeasuring (directly or indirectly) the formation of a complex. Thesample can be obtained directly or indirectly, and can be prepared by amethod suitable for the particular sample and assay format selected.

In particular, tissue samples, e.g., gill tissue samples, can be takenfrom fish after they are anaesthetized with MS-222. The tissue samplesare fixed by immersion in 2% paraformaldehyde in appropriate Ringerssolution corresponding to the osmolality of the fish, washed in Ringers,then frozen in an embedding compound, e.g., O.C.T.™ (Miles, Inc.,Elkahart, Ind., USA) using methylbutane cooled with liquid nitrogen.After cutting 8-10 micron tissue sections with a cryostat, individualsections are subjected to various staining protocols. For example,sections are: 1) blocked with goat serum or serum obtained from the samespecies of fish, 2) incubated with rabbit anti-CaR or anti-SalmoKCaRantiserum, and 3) washed and incubated with peroxidase-conjugatedaffinity-purified goat antirabbit antiserum. The locations of the boundperoxidase-conjugated goat antirabbit antiserum are then visualized bydevelopment of a rose-colored aminoethylcarbazole reaction product.Individual sections are mounted, viewed and photographed by standardlight microscopy techniques. The anti-CaR antiserum used to detect fishSalmoKCaR protein is raised in rabbits using a 23-mer peptidecorresponding to amino acids numbers 214-236 localized in theextracellular domain of the RaKCaR protein. The sequence of the 23-merpeptide is:

-   -   ADDDYGRPGIEKFREEAEERDIC (SEQ ID NO.: 24) A small peptide with        the sequence DDYGRPGIEKFREEAEERDICI (SEQ ID NO.: 25) or    -   ARSRNSADGRSGDDLPC (SEQ ID NO.: 26) can also be used to make        antisera containing antibodies to SalmoKCaRs. Such antibodies        can be monoclonal, polyclonal or chimeric.

Suitable labels can be detected directly, such as radioactive,fluorescent or chemiluminescent labels. They can also be indirectlydetected using labels such as enzyme labels and other antigenic orspecific binding partners like biotin. Examples of such labels includefluorescent labels such as fluorescein, rhodamine, chemiluminescentlabels such as luciferase, radioisotope labels such as ³²P, ¹²⁵I, ¹³¹I,enzyme labels such as horseradish peroxidase, and alkaline phosphatase,β-galactosidase, biotin, avidin, spin labels, magnetic beads and thelike. The detection of antibodies in a complex can also be doneimmunologically with a second antibody which is then detected (e.g., bymeans of a label). Conventional methods or other suitable methods candirectly or indirectly label an antibody. Labeled primary and secondaryantibodies can be obtained commercially or prepared using methods knowto one of skill in the art (see Harlow, E. and D. Lane, 1988,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y.).

Using the immunocytochemistry method, the levels of SalmoKCaR in varioustissues can be detected and examined as to whether they change incomparison to control. Modulated levels or the presence of SalmoKCaRexpression in various tissues, as compared to a control, indicate thatthe fish or the population of fish from which a statisticallysignificant amount of fish were tested, are ready for transfer tofreshwater. A control refers to a level of SalmoKCaR, if any, from afish that is not subjected to the steps of the present invention, e.g.,not subjected to freshwater having a SalmoKCaR modulator and/or not feda NaCl diet. For example, FIGS. 18 and 19 show that fish not subjectedto the present invention had no detectable SalmoKCaR level, whereas fishthat were subjected to the steps of the invention had SalmoKCaR levelsthat were easily detected.

In determining whether compounds are modulators, one can measure changesthat occur in the expression levels of one or more the SalmoKCaR genes,or those that occur in one or more intracellular signal transductionsystems or pathways. A signal transduction pathway is a pathway involvedin the sensing and/or processing of stimuli. In particular, suchpathways are altered by activation of the expressed proteins coded forby a single or combination of nucleic acids of the present invention.

The SalmoKCaR polypeptides can be in the form of a conjugate or a fusionprotein, which can be manufactured by known methods. Fusion proteins canbe manufactured according to known methods of recombinant DNAtechnology. For example, fusion proteins can be expressed from a nucleicacid molecule comprising sequences which code for a biologically activeportion of the SalmoKCaR polypeptide and its fusion partner, for examplea portion of an immunoglobulin molecule. For example, some embodimentscan be produced by the intersection of a nucleic acid encodingimmunoglobulin sequences into a suitable expression vector, phagevector, or other commercially available vectors. The resulting constructcan be introduced into a suitable host cell for expression. Uponexpression, the fusion proteins can be isolated or purified from a cellby means of an affinity matrix. By measurement of the alternations inthe functions of transfected cells occurring as a result of expressionof recombinant SalmoKCaR proteins, either the cells themselves orSalmoKCaR proteins produced from the cells can be utilized in a varietyof screening assays that all have a high degree of utility overscreening methods involving tests on the same PVCR proteins in wholefish.

The SalmoKCaRs can also be assayed by Northern blot analysis of mRNAfrom tissue samples. Northern blot analysis from various shark tissueshas revealed that the highest degree of PVCR expression is in gilltissue, followed by the kidney and the rectal gland. There appear to beat least three distinct mRNA species of about 7 kb, 4.2 kb and 2.6 kb.

The SalmoKCaRs can also be assayed by hybridization, e.g., byhybridizing one of the SalmoKCaR sequences provided herein (e.g., SEQ IDNO: 7,9 or 11) or an oligonucleotide derived from one of the sequences,to a DNA or RNA-containing tissue sample from a fish. Such ahybridization sequence can have a detectable label, e.g., radioactive,fluorescent, etc., attached to allow the detection of hybridizationproduct. Methods for hybridization are well known, and such methods areprovided in U.S. Pat. No. 5,837,490, by Jacobs et al., the entireteachings of which are herein incorporated by reference in theirentirety. The design of the oligonucleotide probe should preferablyfollow these parameters: (a) it should be designed to an area of thesequence which has the fewest ambiguous bases (“N's”), if any, and (b)it should be designed to have a T_(m) of approx. 80° C. (assuming 2° C.for each A or T and 4 degrees for each G or C).

Additionally, the above probes could be used in a kit to identifySalmoKCaR homologs and their expression in various fish tissue. Thepresent invention also encompasses the isolation of SalmoKCaR homologsand their expression in various fish tissues with a kit containingprimers specific for conserved sequences of SalmoKCaR nucleic acids andproteins.

The present invention encompasses detection of SalmoKCaRs with PCRmethods using primers disclosed or derived from sequences describedherein. For example, SalmoKCaRs can be detected by PCR using SEQ ID Nos:13 and 14, as described in Example 6. PCR is the selective amplificationof a target sequence by repeated rounds of nucleic acid replicationutilizing sequence-specific primers and a thermostable polymerase. PCRallows recovery of entire sequences between two ends of known sequence.Methods of PCR are described herein and are known in the art.

In particular, the levels of SalmoKCaR nucleic acid can be determined invarious tissues by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) after isolation of poly A+ RNA from aquatic species. Methods ofPCR and RT-PCR are well characterized in the art (See generally, PCRTechnology: Principles and Applications for DNA Amplification (ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (Eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila et al., Nucleic Acids Res., 19:4967 (1991);Eckert et al., PCR Methods and Applications, 1:17 (1991); PCR (eds.McPherson et al., IRL Press, Oxford); Ausebel, F. M. et al., CurrentProtocols in Molecular Biology, Greene Publishing Assoc. andWiley-Interscience 1987, & Supp. 49, 2000; and U.S. Pat. No. 4,683,202).Briefly, mRNA is extracted from the tissue of interest and reversetranscribed. Subsequently, a PCR reaction is performed withSalmoKCaR-specific primers and the presence of the predicted SalmoKCaRproduct is determined, for example, by agarose gel electrophoresis.Examples of SalmoKCaR-specific primers are SEQ ID NO: 16-21. The productof the RT-PCR reaction that is performed with SalmoKCaR-specific primersis referred to herein as a RT-PCR product. The RT-PCR product caninclude nucleic acid molecules having part or all of the SalmoKCaRsequence. The RT-PCR product can optionally be radioactively labeled andthe presence or amount of SalmoKCaR product can be determined usingautoradiography. Two examples of commercially available fluorescentprobes that can be used in such an assay are Molecular Beacons(Stratagene) and Taqman® (Applied Biosystems). Alternative methods oflabeling and quantifying the RT-PCR product are well known to one ofskill in the art (see Ausebel, F. M. et al., Current Protocols inMolecular Biology, Greene Publishing Assoc. and Wiley-Interscience 1987,& Supp. 49, 2000. Poly A+ RNA can be isolated from any tissue whichcontains at least one SalmoKCaR by standard methods. Such tissuesinclude, for example, gill, nasal lamellae, urinary bladder, kidney,intestine, stomach, liver and brain.

Hence, the present invention includes kits for the detection ofSalmoKCaR or the quantification of SalmoKCaR having either antibodiesspecific for SalmoKCaR or a portion thereof, or a nucleic acid sequencethat can hybridize to the nucleic acid of SalmoKCaR.

Transgenic Fish

Alterations in the expression or sensitivity of SalmoKCaRs could also beaccomplished by introduction of a suitable transgene. Suitabletransgenes would include either the SalmoKCaR genes itself or modifiergenes that would directly or indirectly influence SalmoKCaR geneexpression. Methods for successful introduction, selection andexpression of the transgene in fish oocytes, embryos and adults aredescribed in Chen, T T et al., Transgenic Fish, Trends in Biotechnology8:209-215 (1990).

The present invention is further and more specifically illustrated bythe following Examples, which are not intended to be limiting in anyway.

EXEMPLIFICATION

The following examples refer to Process I and Process II throughout.Process I is also referred to herein as “SUPERSMOLT™ I Process” or “APSProcess I.” APS stands for “AquaBio Products Sciences®, L.L.C.” A“Process I” fish or smolt refers to a fish or smolt that has undergonethe steps of Process I. A Process I smolt is also referred to as a“SUPERSMOLT™ I” or an “APS Process I ” smolt. Likewise, Process II isalso referred to herein as “SUPERSMOLT™ II Process” or “Process II.” A“Process II” fish or smolt refers to a fish or smolt that has undergonethe steps of Process II. A Process II smolt is also referred to as a“SUPERSMOLT™ II” or an “APS Process II” smolt.

Process I: Pre-adult anadromous fish (this includes both commerciallyproduced S0, S1 or S2 smolts as well as smaller parr/smolt fish) areexposed to or maintained in freshwater containing either 2.0-10.0 mMCalcium and 0.5-10.0 mM Magnesium ions. This water is prepared byaddition of calcium carbonate and/or chloride and magnesium chloride tothe freshwater. Fish are fed with feed pellets containing 7%(weight/weight) NaCl. Fish are exposed to or maintained in this regimenof water mixture and feed for a total of 30-45 days, using standardhatchery care techniques. Water temperatures vary between 10-16° C. Fishare exposed to a constant photoperiod for the duration of Process I. Afluorescent light is used for the photoperiod.

Process II: Pre-adult anadromous fish (this includes both commerciallyproduced S0, S1 or S2 smolts as well as smaller parr/smolt fish) areexposed to or maintained in freshwater containing 2.0-10.0 mM Calciumand 0.5-10.0 mM Magnesium ions. This water is prepared by addition ofcalcium carbonate and/or chloride and magnesium chloride to thefreshwater. Fish are fed with feed pellets containing 7% (weight/weight)NaCl and either 2 gm or 4 gm of L-Tryptophan per kg of feed. Fish areexposed to or maintained in this regimen of water mixture and feed for atotal of 30-45 days using standard hatchery care techniques. Watertemperatures vary between 10-16° C. Fish are exposed to a constantphotoperiod for the duration of Process II. A fluorescent light is usedfor the photoperiod.

EXAMPLE 1 Molecular Cloning of Shark Kidney Calcium Receptor RelatedProtein (SKCaR)

A shark λZAP cDNA library was manufactured using standard commerciallyavailable reagents with cDNA synthesized from poly A+ RNA isolated fromshark kidney tissue as described and published in Siner et al. Am. J.Physiol. 270:C372-C381, 1996. The shark cDNA library was plated andresulting phage plaques screened using a ³²P-labeled full length ratkidney CaR (RaKCaR) cDNA probe under intermediate stringency conditions(0.5×SSC, 0.1% SDS, 50° C.). Individual positive plaques were identifiedby autoradiography, isolated and rescued using phagemid infections totransfer cDNA to KS Bluescript vector. The complete nucleotide sequence,FIG. 1, (SEQ ID NO: 1) of the 4.1 kb shark kidney PVCR related protein(SKCaR) clone was obtained using commercially available automatedsequencing service that performs nucleotide sequencing using the dideoxychain termination technique. The deduced amino acid sequence (SEQ ID NO:2) is shown in FIG. 1. Northern analyses were performed as described inSiner et. al. Am. J. Physiol. 270:C372-C381, 1996. The SKCAR nucleotidesequence was compared to others CaRs using commercially availablenucleotide and protein database services including GENBANK and SWISSPIR.

EXAMPLE 2 Expression/Activation Studies of SKCaR in Human EmbryonicKidney (HEK) Cells

PVCRs serve as salinity sensors in fish. These receptors are localizedto the apical membranes of various cells within the fish's body (e.g.,in the gills, intestine, kidney) that are known to be responsible forosmoregulation. A full-length cation receptor (CaR, also referred to as“PVCR”) from the dogfish shark has been expressed in human HEK cells.This receptor was shown to respond to alterations in ionic compositionsof NaCl, Ca2+ and Mg2+ in extracellular fluid bathing the HEK cells. Theionic concentrations encompassed the range which includes the transitionfrom freshwater to seawater. Expression of PVCR mRNA is also increasedin fish after their transfer from freshwater to seawater, and ismodulated by PVCR agonists. Partial genomic clones of PVCRs have alsobeen isolated from other fish species, including winter and summerflounder and lumpfish, by using nucleic acid amplification withdegenerate primers.

In particular, the following was shown:

1. SKCaR encodes a functional ion receptor that is sensitive to bothMg2+ and Ca2+ as well as alterations in NaCl.

2. SKCaR's sensitivity to Ca2+, Mg2+ and NaCl occur in the range that isfound in marine enviromnents and is consistent with SKCaRs role as asalinity sensor.

3. SKCaR's sensitivity to Mg2+ is further modulated by Ca2+ such thatSKCaR is capable to sensing various combinations of divalent andmonovalent cations in seawater and freshwater. These data can be used todesign novel electrolyte solutions to maintain fish in salinitiesdifferent from those present in their natural environment.

SKCaR cDNA was ligated into the mammalian expression vector PCDNA II andtransfected into HEK cells using standard techniques. The presence ofSKCaR protein in transfected cells was verified by western blotting.Activation of SKCaR by extracellular Ca2+, Mg2+ or NaCl was quantifiedusing a well characterized FURA 2 based assay where increases inintracellular Ca2+ produced by SKCaR activation are detected usingmethodology published previously by Bai, M., S. Quinn, S. Trvedi, O.Kifor, S. H. S. Pearce, M. R. Pollack, K. Krapcho, S. C. Hebert and E.M. Brown. Expression and characterization of inactivating and activatingmutations in the human Ca2+-sensing receptor. J. Biol. Chem.,32:19537-19545 (1996); and expressed as % normalized intracellularcalcium response to receptor activation.

SKCaR is a functional extracellular Ca2+ sensor where its sensitivity ismodulated by alterations in extracellular NaCl concentrations. As shownin FIG. 2, SKCaR is activated by increasing concentrations ofextracellular Ca2+ where half maximal activation of SKCaR ranges between1-15 mM depending on the extracellular concentration of NaCl. These arethe exact ranges of Ca2+ (1-10 mM present in marine estuarian areas).Note that increasing concentrations of NaCl reduce the sensitivity ofSKCaR to Ca2+. This alteration in SKCaR sensitivity to Ca2+ was notobserved after addition of an amount of sucrose sufficient to alter theosmolality of the extracellular medium. This control experiment shows itis not alterations in cell osmolality effecting the changes observed.

The half maximal activation (EC₅₀) by Ca2+ for SKCaR is reduced inincreased concentrations of extracellular NaCl. See FIG. 4. The EC₅₀ fordata shown on FIG. 4 is displayed as a function of increasingextracellular NaCl concentrations. Note the EC₅₀ for Ca2+ increases fromless than 5 mM to approximately 18 mM as extracellular NaClconcentrations increase from 50 mM to 550 mM.

SKCaR is a functional extracellular Mg2+ sensor where its sensitivity ismodulated by alterations in extracellular NaCl concentrations. As shownin FIG. 3, SKCaR is activated in the range of 5-40 mM extracellular Mg2+and is modulated in a manner similar to that shown in FIGS. 2 and 4 byincreasing concentrations of extracellular NaCl. Similarly, thisalteration in SKCaR sensitivity to Ca2+ was not observed after additionof an amount of sucrose sufficient to alter the osmolality of theextracellular medium.

The half maximal activation (EC₅₀) by Mg2+ for SKCaR is reduced inincreased concentrations of extracellular NaCl. See FIG. 5. The EC₅₀ fordata shown on FIG. 5 is displayed as a function of increasingextracellular NaCl concentrations. Note the EC₅₀ for Mg2+ increases fromless than 20 mM to approximately 80 mM as extracellular NaClconcentrations increase from 50 mM to 550 mM.

Addition of 3mM Ca2+ alters the sensitivity of SKCaR to Mg2+ and NaCl.See FIG. 6. The EC₅₀ for Mg2+ of SKCaR is modulated by increasingconcentrations of NaCl as shown both in this FIG. 6 and in FIG. 5.Addition of 3 mM Ca2+ to the extracellular solution alters thesensitivity characteristics of SKCaR as shown. Note the 3 mM Ca2+increases the sensitivity of SKCaR to Mg2+ as a function ofextracellular NaCl concentrations.

This method was also used to isolate partial genomic clones of PVCRs forAtlantic salmon and other species such as Arctic char and rainbow trout,as described herein. FIGS. 16A-D show the amino acid sequences andalignment for the PVCRs from three full length Atlantic salmon clones(SalmoKCar #1, #2, and #3) relative to the PVCR from the kidney of thedogfish shark (Squalus acanthias) (SKCaR) and human parathyroid calciumreceptor (HuPCaR).

EXAMPLE 3 Defining Salinity Limits as an Assay to Identify Fish withEnhanced Salinity Responsive and Altered PVCR Function

Both anadromous fish (Atlantic salmon, trout and Arctic char) andeuryhaline fish (flounders, alewives, eels) traverse from freshwater toseawater environments and back again as part of their lifecycles in thenatural environment. To successful accomplish this result; both types offish have to undergo similar physiological changes including alterationsin their urine output, altering water intake and water absorption. Insome cases, naturally occurring mutations to PVCR would provide foraltered salinity adaptation capabilities that would have significantvalue for both commercial and environmental restoration uses. Forexample, identification of selective traits associated with PVCRmediated salinity responses might allow identification of new strains offish for commercial aquaculture. Similarly, identification of selectedenvironmental parameters from a host of natural and man made variablesthat are the most important to improve the survival and successfulrestocking and/or ocean ranching of either wild Atlantic salmon orwinter flounder would also be of great utility. To permit theidentification of individual fish possessing enhanced salinityresponsive characteristics, assays must be designed that enable thesefish to survive while others not possessing these characteristics willeither die or perform poorly. As described below, such assays would takeadvantage of the ability of these anadromous and euryhaline fish towithstand a wide range of salinities. Fish that were identified usingsuch assays would then be propagated in breeding-selection programs.

Winter and Summer Flounder can be grown and maintained in recyclingwater systems. Groups of both winter (Pleuronectes americanus) andsummer (Paralichthus dentalus) flounder were maintained in multiplemodular recycling water system units that are composed of a single 1meter fish tank maintained by a 1 meter biofilter tank located directlyabove it. The upper tank of each unit contains 168 sq. ft. of biofiltersurface area that will support a maximum of 31 lbs of flounder, whilemaintaining optimal water purity and oxygenation conditions. Each unitis equipped with its own pump and temperature regulator apparatus. Boththe temperature and photo-period of each unit can be independentlyregulated using black plastic curtains that partition each tank off fromits neighbor. The inventors have a total of 12 independent modular unitsthat permit 3 experiments each with 4 variables to be performedsimultaneously. Using this experimental system, the following data havebeen obtained.

Salinity survival limits for winter and summer flounder with a constantratio of divalent and monovalent ions were determined. The survivallimit of both winter and summer flounder in waters of salinities greaterthan normal seawater (10 mM Ca2+, 50 mM Mg2+ and 450 mM NaCl) is watercontaining twice (20 mM Ca2+, 50 mM Mg2+ and 900 mM NaCl) the normalconcentrations of ions present in normal seawater. In contrast, thesurvival limit of both winter and summer flounder in waters of salinityless than normal seawater is 10% seawater (1 mM Ca2+, 5 mM Mg2+ and 45mM NaCl).

Use of a fully recycling water system permits growth of flounder atvastly different salinities. Groups of flounder (n=10) were adapted overa 15 day interval and maintained at either low salinity (LS) (e.g., at10% normal seawater), normal seawater (NS) or hypersalinity (HS) (e.g.,2X seawater) for intervals of 3 months, under otherwise identicalconditions. Survival among the 3 groups were comparable (all greaterthan 80%) and there were no differences in the electrolyte content oftheir respective sera.

EXAMPLE 4 Isolation of Partial Atlantic Salmon PVCRs

A partial PVCR gene of Atlantic Salmon was isolated as follows:sequences of shark kidney calcium receptor together with the nucleotidesequence of mammalian calcium receptors were used to design degenerateoligonucleotide primers, dSK-F3 (SEQ ID NO: 13) and dSK-R4 (SEQ ID NO:14), to highly conserved regions in the transmembrane domain ofpolyvalent cation receptor proteins using standard methodologies (See GM Preston, Polymerase chain reaction with degenerate oligonucleotideprimers to clone gene family members, Methods in Mol. Biol. Vol. 58Edited by A. Harwood, Humana Press, pages 303-312, 1993). Using theseprimers, genomic DNA from the above species was amplified using standardPCR methodology. The PCR product (653 nt) was then purified by agarosegel electrophoresis and ligated into appropriate plasmid vector that wasthen transformed into a bacterial strain. After growth in liquid media,vectors and inserts are purified using standard techniques, analyzed byrestriction enzyme analysis and sequenced. Using this methodology, atotal of 8 nucleotide sequences from 8 fish species including AtlanticSalmon were amplified. Each clone is 594 nt (with-out primer sequences)and encodes a 197 amino acid sequence which corresponds to the conservedtransmembrane domain of the calcium receptors.

Atlantic salmon partial PVCR nucleic acid sequence (SEQ ID NO: 3) iscomposed of 594 nucleotides (nt) containing an open reading frameencoding 197 amino acids (SEQ ID NO: 4) (FIG. 7).

Primer Sequences for PCR of PVCR Clones: dSK-F3 (SEQ ID NO:13) 5′-TGTCKT GGA CGG AGC CCT TYG GRA TCG C-3′ dSK-R4 (SEQ ID NO:14) 5′-GGC KGGRAT GAA RGA KAT CCA RAC RAT GAA G-3′I=deoxyinosine, N=A+C+T+G, R=A+G, Y=C+T, M=A+C, K=T+G, S=C+G, W=A+T,H=A+T+C, B=T+C+G, D=A+T+G, V=A+C+G; Product from amplification=653 nt

EXAMPLE 5 Molecular Cloning of a Second Partial Atlantic Salmon PVCR

A second Atlantic salmon partial PVCR was isolated, as described herein.An Atlantic salmon λZAP cDNA library was manufactured using standardcommercially available reagents with cDNA synthesized from poly A+ RNAisolated from Atlantic salmon intestine tissue according tomanufacturers instructions (Stratagene, La Jolla, Calif.) and screenedusing the Atlantic salmon PCR product as a probe. A partial Atlanticsalmon PVCR cDNA (SEQ ID NO: 5) is composed of 2021 nucleotides (nt)(FIG. 8A) containing an open reading frame encoding 388 amino acids (SEQID NO: 6) (FIG. 8B). The open reading frame encoded by SEQ ID NO: 5begins at nucleotide position 87.

EXAMPLE 6 Molecular Cloning of 3 Full Length cDNA Clones from Kidney ofAtlantic Salmon (Salmo Salar) and Determination of their Tissue SpecificExpression in Various Salmon Tissues Modulated by Water Salinity

In Example 5, a homology based approach was used to screen cDNAlibraries under moderate stringency conditions to obtain a full lengthshark kidney PVCR clone (SKCaR). Using sequence information derived fromExamples 4 and 5, both nucleotide (nt) and antibody probes were designedto detect PVCRs in other fish species. Using degenerate primers whosesequence was derived from knowledge of the nt sequence of SKCaR, PCR wasutilized to amplify a series of genomic and cDNA (RT-PCR) sequences thatcontain partial nt and putative protein sequences of PVCRs from multiplefish including Atlantic salmon. See Examples 1, 4, and 5.

The data described in this Example show that the nt and putative proteinsequences of 3 PVCR transcripts from Atlantic salmon kidney wereisolated and characterized. Additionally, their tissue specificexpression and modulation of tissue expression levels by alterations inwater salinity were determined. This Example is divided into 2 parts: 1)isolation and sequence of 3 full length PVCR clones from salmon kidney(SalmoKCar#1 (SEQ ID NO: 7), SalmoKCar#2 (SEQ ID NO: 9) and SalmoKCar#3(SEQ ID NO: 11)) and 2) use of RT-PCR analysis with degenerate and clonespecific SalmoKCaR PCR primers to determine the tissue specificexpression of these 3 transcripts in seawater vs. freshwater as well asthe SuperSmolt™ process. Taken together, these data provide theframework for achieving a fundamental understanding of both PVCRs insalmonids as well as the their roles in the SuperSmolt™ process.

Part 1. Isolation and Sequence of 3 Full Length PVCR Clones from SalmonKidney:

Materials and Methods: Total RNA was purified with Stat 60 reagent(Teltest B Friendswood, Tex.) and poly A⁺ purified with the MicroFastTrack Kit (Invitrogen, Carlsbad, Calif.). cDNA was then synthesizedand fractionated whereby selected fractions were ligated and packaged asλZAP libraries (Stratagene, La Jolla, Calif.). Library phage were thenplated and duplicate filter lifts performed that were screened underhigh stringency (0.1×SSC, 0.1% SDS @ 55° C.) with a ³²P-labeled(RadPrime Kit, Invitrogen, Carlsbad, Calif.) genomic fragment ofAtlantic salmon PVCR (653 nt sequence) amplified using protocols andreagents described in Examples 1, 4 and 5. Primary positive plaques werepurified, excised and sequenced using commercial sequencing services (U.of Maine, Orono, Me.) and their sequences compared with those of otherPVCRs using BLAST. (National Library of Medicine, Bethesda, Md.).

Results: A total of seven cDNA clones containing PVCR sequences wereidentified and purified from Atlantic Salmon kidney and intestinelibraries. A total of three of the seven contain full length codingsequences for PVCR proteins together with 5′ and 3′ regulatory elements.For convenience, these clones are designated Salmo salar Kidney PVCRs(SalmoKCaRs) #1, #2 and #3 and their aligned nt and putative proteinsequences are shown in FIGS. 12 and 13, respectively. The remaining 4positive clones were partial PVCR clones very nearly identical to these3 full-length SalmoKCaR clones. Comparison of the different nt sequencesof these 3 clones reveals the following similarities and differences:

-   -   The SalmoKCaR #1 nucleic acid sequence (SEQ ID NO: 7) consists        of 3941 nts of 5′ and 3′ regulatory elements together with        full-length coding sequence for a 941 AA PVCR protein (SEQ ID        NO: 8). See FIGS. 9A-E. The calculated molecular mass of this        protein is 106,125 Daltons.    -   The SalmoKCaR #2 nucleic acid sequence (SEQ ID NO: 9) consists        of 4031 nts of 5′ and 3′ regulatory elements together with        full-length coding sequence for a 941 AA PVCR protein (SEQ ID        NO: 10). See FIGS. 10A-E. The calculated molecular mass of this        protein is 106,180 Daltons.    -   The SalmoKCaR #3 nucleic acid sequence (SEQ ID NO: 11) consists        of 3824 nts of 5′ and 3′ regulatory elements together with        full-length coding sequence for a 850 AA PVCR protein (SEQ ID        NO: 12). See FIGS. 11A-D. The calculated molecular mass of this        protein is 96,538 Daltons.

FIGS. 12A-L and 13A-C show an alignment of between the two partialsequences of Atlantic Salmon PVCRs isolated and the 3 full length clonesfor both the nucleic acid and amino acid sequences, respectively. Onepartial nucleic acid sequence of an Atlantic Salmon PVCR, SEQ ID NO: 3,can be found in all three SalmoKCaR nucleic acid sequences between nt1979 and 2572; nt 2069 and 2662; and nt 1980 and 2573 of SEQ ID NO: 7,9, and 11, respectively. The second partial Atlantic Salmon clone, SEQID NO: 5, can also be found in all three SalmoKCaR nucleic acidsequences: between nt 1753 and 3773; 1843 and 3863, and 1754 and 3616 ofSEQ ID NO: 7, 9, and 11, respectively. Similarly, the amino acidsequence of SEQ ID NO: 4 is found between aa 601 and 797 of each of SEQID NO: 8, 10, and 12. The amino acid sequence of the second AtlanticSalmon Clone, SEQ ID NO: 6, is found in each of the polypeptides:between aa 554 and 941 of SEQ ID NO: 8; between aa 554 and 941 of SEQ IDNO: 10; and between aa 554 and 850 of SEQ ID NO: 12. Note that the aminoacid sequence of SEQ ID NO: 6 extends 91 aa past the end of SEQ ID NO:12.

Additional differences between the partial Atlantic salmon PVCR (SEQ IDNO: 5) and full length PVCR (SEQ ID NO: 7, 9, or 11) include: nt 1-112do not align with any corresponding sequence in SEQ ID NO: 7, 9, or 11.There are also 4 single nt base pair substitutions that are present inSEQ ID NO: 5 that are different than corresponding nt in full length SEQID NO: 7, 9, or 11. These include:

-   -   nt 1893 change from G to A    -   nt 1970 change from G to A    -   nt 1973 change from G to A    -   nt 2001 change from G to A.

Table 1 compares the overall % identity of nucleotides (nt) between cDNAclones that contain the SalmoKCaRs #1,2 and 3 vs. shark kidney calciumreceptor (SKCaR containing 4079 nts) or human parathyroid CaR (HuPCaRcontaining 3783 nts). Note that all 3 SalmoKCaR clones possessapproximately a 56-57% nt identity to SKCaR and an approximately 50-55%nt identity to HuPCaR. However, in spite of the rather low overall % ntidentity between the 3 SalmoKCaR clones and SKCaR, all 3 full lengthSalmoKCaR clones hybridize to full length SKCaR clone under highstringency conditions (0.5×SSC, 0.1% SDS @ 65° C.) (See FIG. 14).

The percentage identities between the aligned nucleotide sequences ofthe 3 full length SalmoKCaR clones (SEQ ID NO: 7, 9, 11) include:

A total of 99.8% of the nt of SEQ ID NO: 7 are identical to those ofcorresponding SEQ ID NO: 9. A total of 97.6% of the nt of SEQ ID NO: 9are identical to those corresponding nt of SEQ ID NO: 7.

A total of 93.6% of the nt of SEQ ID NO: 9 are identical to thosecorresponding nt of SEQ ID NO: 11. A total of 98.7% of the nt of SEQ IDNO: 11 are identical to the corresponding nt present in SEQ ID NO: 9.

A total of 95.8% of the nt of SEQ ID NO: 7 are identical to thecorresponding nt of SEQ ID NO: 11. A total of 98.7% of the nt of SEQ IDNO: 11 are identical to those corresponding in SEQ ID NO: 7. TABLE 1Comparison of the % nucleotide (nt) identity of the complete nt sequenceof 3 SalmoKCaR clones #1, #2 and #3 (including 5′ and 3′ regulatoryelements vs. either the SKCaR clone or the clone HuPCaR clone. %NUCLEOTIDE IDENTITY SalmoKCaR ^(#)1 SalmoKCaR ^(#)2 SalmoKCaR ^(#)3SKCaR vs. 56.2 56.5 57.2 HuPCaR vs. 55.0 54.9 50.9

Table 2 compares both the overall and domain-specific percent amino acid(% AA) identity for each of the SalmoKCaR clones vs. shark kidney PVCR(SKCaR-upper half) and human parathyroid CaR (HuPCaR-lower half). Whencompared to SKCaR, all 3 SalmoKCaR proteins possess approximately a63-68% overall AA identity to SKCaR. However, their domain-specificidentities show significant degrees of variation with the carboxylterminal domain of the SalmoKCaR 3 being the most widely divergent. Notsurprisingly, comparisons between the 3 SalmoKCaR proteins vs. HuPCaRreveal that the 7 transmembrane region possesses the highest degree ofhomology followed by the extracellular domain and finally theintracellular carboxy terminal domain.

The percentage identities between the aligned amino acid sequences ofthe 3 full length SalmoKCaR clones (SEQ ID NO: 8, 10, or 12) include:

A total of 99.9% of the aa of SEQ ID NO: 8 are identical to thosecorresponding aas in SEQ ID NO: 10. A total of 99.9% of the aa of SEQ IDNO: 10 are identical to corresponding aa in SEQ ID NO: 8.

A total of 89.5% of the aa of SEQ ID NO: 10 are identical to thosecorresponding aas in SEQ ID NO: 12. A total of 99.1% of the aa of SEQ IDNO: 12 are identical to those corresponding aa in SEQ ID NO: 10.

A total of 89.6% of the aa of SEQ ID NO: 8 are identical to thosecorresponding aas in SEQ ID NO: 12. A total of 99.2% of the aa of SEQ IDNO: 12 are identical to those corresponding aa of SEQ ID NO: 8. TABLE 2Comparison of % amino acid (AA) identities of 3 SalmoKCaR proteins vs.AA sequence of shark kidney CaR (SKCaR-Upper Half) and human parathyroidCaR (HuPCaR-Lower Half). SalmoKCaR SalmoKCaR SalmoKCaR ^(#)1 ^(#)2 ^(#)3% AA Identity to SKCaR Overall Protein 68.4 68.3 63.3 Domains N-terminalExtracellular Ion 70.0 69.8 70.0 Binding Domain 7 Transmembrane Region87.2 87.2 86.4 Carboxyl Terminal 31.8 31.8 0.0 IntraCellular Domain % AAIdentity to HuPCaR Overall Protein 66.3 66.3 61.4 Domains N-terminalExtracellular Ion 71.9 71.9 72.1 Binding Domain 7 Transmembrane Region89.2 89.2 88.4 Carboxyl Terminal 24.1 24.1 0 IntraCellular Domain

FIG. 14 shows all 3 unique SalmoKCaR clones hybridize to full lengthshark kidney CaR (SKCaR) under high stringency conditions (0.5×SSC, 0.1%SDS @ 65° C.). Representative autoradiogram of Southern blot was exposedfor 30 min.

Site directed mutagenesis studies of mammalian CaRs, notably HuPCaR,have identified AAs that are particularly important in the variousfunctions of CaRs. Cysteine AAs at AA#101 and AA#236 mediatedimerization of HuPCaR. HuPCaR and native CaRs in rat kidney existprimarily as dimers within the cell membrane where disulfidebond-mediated dimerization is required for normal agonist-mediated CaRactivation. All 3 SalmoKCaRs possess Cys at AAs corresponding to HuPCaRAA#101 and AA#236 and presumably functions as dimers in a manner similarto mammalian CaRs.

Nucleotide Sequence Differences in the 5′ and 3′ Untranslated Regions orUTRs of SalmoKCaRs #1, #2 and #3:

FIG. 15 displays the aligned nucleotide sequences of SalmoKCaR clones#1, #2, and 3. As compared to SalmoKCaR #1 and #3, SalmoKCaR #2possesses an 89 nt insert in its 5′ UTR. Differences between the 3′ UTRsof the 3 SalmoKCaRs include a 36 nt insert just prior to the poly A tailin SalmoKCaR #3 as well as other single nt differences listed belowwhere each difference is compared to the 2 other SalmoKCaR clones:

-   -   SalmoKCaR#1: nt 3660 A to G; nt 3739 A to G; nt 3745 A to G    -   SalmoKCaR #2: nt 3837 A to G; nt 3862 A to G    -   SalmoKCaR #3: nt 3472 A to G; nt 3487 A to G; nt 3564 A to G; nt        3568 G to A; nt 3603 A to G; nt 3786 A to C.        Although the functional significance of each of these nt        differences in the 5′ or 3′ UTRs is unknown at the present time,        each nt difference either individually or in combinations could        represent a means for controlling either the stability or        processing of the RNA transcript or its translation into each of        the 3 SalmoKCaR proteins.        Sequence Differences in the Coding Regions of SalmoKCaRs #1, #2        and #3:

FIG. 16 displays the aligned AA sequences of SalmoKCaRs #1, #2 and #3 aswell as the Shark SKCaR protein and HuPCaR proteins. As compared toSalmoKCaR #1, SalmoKCaR #2 possesses 2 different AA's present at AA#257and AA#941 of its AA sequence. In contrast to SalmoKCaR #1 thatpossesses an Asp in AA#257, SalmoKCaR #2 possesses a Gly. The negativecharge in this location may be important since both SKCaR and Fugu PVCRpossess Asp at #257 while the mammalian CaRs, HuPCaR and RaKCaR possessa Glu. SalmoKCaR #3 also contains a Asp at AA#257.

At AA #443, SalmoKCaR #1 and #2 both possess a Leu whereas SalmoKCaR #3contains a Phe. The conserved hydrophobic nature of the AA at thisposition appears to be important since Fugu PVCR also contains a Leuwhereas SKCaR contains an Ile. As compared to SalmoKCaRs #1 or 2,SalmoKCaR #3 possesses a truncated carboxyl terminus as described below.

Sequence Differences in the Coding Regions of SalmoKCaRs #1, #2 and #3as Compared to Mammalian CaRs.

The putative AA sequences of SalmoKCaR #1, #2 and #3 proteins possessmultiple differences in AAs at various positions throughout theirextracellular, 7 transmembrane and carboxyl terminal domains whencompared to mammalian CaRs such as HuPCaR (see aligned differences withHuPCaR in FIG. 16). While many of the differences between SalmoKCaRspecies and HuPCaR are conserved substitutions that preserve the overallnet charge or hydrophobicity characteristics at that specific positionin the PVCR protein, other substitutions may have functionalconsequences as based on previous structure-functional studies ofmammalian CaRs. The actual functional consequences of these AAdifferences in SalmoKCaR proteins await expression studies by MariCal.

Differences between SalmoKCaR proteins vs. mammalian and other fishPVCRs include:

-   -   All 3 SalmoKCaRs possess a deletion of 15 AA's beginning at AA        #369 as compared to either HuPCaR or RaKCaR. Fugu PVCR also        exhibits a 19 AA deletion at the same location. In contrast,        SKCaR does not exhibit any deletion in this area and thus is        more similar to mammalian CaRs as compared to either SalmoKCaR        or Fugu in this regard.    -   Another notable difference between SalmoKCaRs vs. mammalian CaRs        and SKCaR is differences in AA #227 where mutagenesis studies        have identified the presence of the positively charged Arg as        important in CaR sensitivity since its alteration in HuPCaR to a        Leu results in over a 2 fold reduction in EC₅₀ Ca²⁺ from 4.0 mM        to 9.3 mM but not Gd³⁺ sensitivity. In contrast to mammalian        CaRs and SKCaR, all 3 SalmoKCaRs possess a negatively charged        Glu at AA#227. Fugu PVCR also exhibits the same Glu at AA#227.        Interestingly, the AA sequence immediately following AA#227 is        Glu-Glu-Ala in the mammalian HuPCaR and elasmobranch SKCaR        whereas it is Lys-Glu-Met in all 3 SalmoKCaRs and Fugu.    -   Lastly, all 3 SalmoKCaR clones as well as Fugu possess an in        frame deletion of a single AA at position #757 (between TM4        and 5) as compared to either mammalian CaRs or SKCaR.    -   SalmoKCaR #3 possesses a truncated carboxyl terminal domain as        compared to either SalmoKCaRs #1 or #2. The number of AA that        comprise the carboxyl terminal domains of the 3 SalmoKCaRs are        different and include: SalmoKCaR #1-96 AA; SalmoKCaR #2-97 AA        and SalmoKCaR #3-5 AA. Reduction in the 91-92AA's in SalmoKCaR        #3 vs. SalmoKCaRs #1 or #2 would reduce its estimated molecular        mass by 9,600 Daltons.

Studies from multiple site directed mutagenesis studies of HuPCaR revealthat alterations to the structure of the carboxyl terminal domain ofPVCRs have profound effects on their function and sensitivity to ligandssuch as Ca²⁺ and Mg²⁺. Various truncations of the carboxyl terminaldomain of HuPCaR have highlighted the importance of HuPCaR AAs #860-910.Truncation of the carboxyl terminal domain of HuPCaR to AAs less thanAA#870 produced either an inactive receptor or a modified HuPCaR with amarked decrease in its affinity for extracellular Ca²⁺ as well as adecrease in the apparent cooperativity of Ca²⁺ dependent activation.While the exact functional characteristics of SalmoKCaR #3 remain to bedetermined using similar HEK transfection studies, these data derivedfrom HuPCaR mutagenesis studies suggest that SalmoKCaR #3 protein iseither inactive or exhibits a greatly reduced functional affinity forCa²⁺. Significant expression of SalmoKCaR #3 together with otherSalmoKCaRs #1 or #2 could result in an overall reduction in the responseto extracellular Ca2+ due to so called dominant negative effects. Thesedominant negative effects could occur where SalmoKCaR#3 reduces theoverall sensitivity of cells to Ca²⁺ via combinations between SalmoKCaR#3 and SalmoKCaR #1/#2 to reduce the sensitivity of the latter PVCRs viacooperative interactions (dimers and higher oligomers) with them.

Certain mutagenesis studies also highlight the importance of theThreonine AA at AA#888 in mediation of HuPCaR's sensitivity to Ca2+ andnormal signal transduction. FIG. 16 shows that AA #888 is a Thr in allwild type CaR and PVCR proteins including HuPCaR, RaKCaR, SKCaR, BoPCaRand SalmoKCaR #1 and #2. SalmoKCaR #3 is missing Thr #888 because of itstruncated tail. Of interest is also the presence of consensus sites forreceptor kinase phosphorylation (Ser-Ser-Ser) that are present atAA#907-909 in HuPCaR, RaKCaR, SKCaR BoPCaR and SalmoKCaR #1 and #2. Incontrast, Fugu PVCR possesses an Asn at AA#908 that would render itssite nonrecongizable to protein kinases. A similar protein kinase sitealso appears in the region of AA#918-921 where HuPCaR, RaKCaR and BoPCaRpossess a Ser-Ser-Ser motif. In contrast, SKCaR possesses an inactivesite due to its sequence of Ala-Ser-Ser. Fugu PVCR and SalmoKCaR #1 and#2 also have intact Ser-Ser-Ser motifs at position AA #918-920 or#919-921. The exact functional significance of these Ser-Ser-Ser sitespossessed by SalmoKCaR#1 and #2 await expression studies by MariCal.

The Presence of Multiple Differences in the Nucleotide and PutativeProtein Sequences of SalmoKCaR Clones #1-#3 Strongly Suggest thePresence of Multiple PVCR Genes Within Atlantic Salmon:

Recent studies in rainbow trout provide direct evidence of the existenceof multiple genes encoding two different forms of a specific type ofprotein, each of which are differentially expressed in specific tissuesof trout. These proteins are aryl hydrocarbon receptor Type 2 (AhRs).Detailed studies on AhRs have shown the presence of 2 functional genesthat produce different closely related AhR proteins, “Two forms of arylhydrocarbon receptor type 2 in rainbow trout (Oncorhynchus mykiss),” byAbnet, C. C., et al., J. of Biological Chemistry 274: 15159-15166,(1999). These two proteins are differentially expressed in varioustissues where they perform closely related but distinct functions.

The presence of single nucleotide substitutions together with specificlarge scale alterations in the sequence of SalmoKCaR clones #1-3including the gapping of large numbers of nucleotides and alterations inreading frame of the resulting SalmoKCaR transcript are not readilyexplainable on the basis of differential splicing of RNA transcriptsderived from a single gene, or perhaps some complex process wheredifferent alleles of a single gene are present in salmon. Alternatively,these data suggest that there are multiple PVCR genes present inAtlantic salmon that work in concert to enable Atlantic salmon andlikely other salmonids to carry out their lifecycle stages that includehatching as well as development of larval and juvenile phases infreshwater followed by smoltification and migration into seawater with asubsequent return to freshwater for spawning.

Detailed studies in mammals including mice and humans show the presenceof a single functional PVCR gene. However, multiple published reportsprovide support for the possibility that multiple PVCR genes exist infish, while only a single functional PVCR gene exists in mammalsincluding humans. Support for multiple PVCR genes is provided bydetailed studies of well characterized genes that have demonstrated thatteleost fish including salmonids possess multiple sets of duplicatedgenes as compared to mammals. These duplicated genes have arisen as aresult of either genomic duplication events occurring early in theevolutionary history of fishes with subsequent gene drop out or via morerecent selective duplication of genes or some combination of both.Moreover, it is widely acknowledged that salmonids are polyploid withrespect to other teleost fish and have undergone an additional genomeduplication. This additional genomic duplication further heightens thepossibility that multiple functional PVCR genes exist in salmonidsparticularly Atlantic salmon.

If the products of a duplicated gene are not important in thedevelopment, growth or maintenance of an organism, the nonfunctionalgene accumulates natural mutations and is either inactivated becoming apseudogene or lost from the genome altogether. However, multiple authorshave provided evidence that preservation of duplicated genes likelyinvolves changes in the developmental or tissue specific expressionpattern of the duplicated vs. original gene or formation of a newfunctional gene protein product that would interact with the originalgene product in novel ways. (See AhR data above). These data providesupport for the possible roles of SalmoKCaR transcripts #1-3 as eitherdifferentially expressed in various tissues of Atlantic salmon as wellas SalmoKCaR #3 exerting a dominant negative effect on the remainingfunctional SalmoKCaR proteins. As discussed below, such interactionsamongst SalmoKCaR transcripts would provide Atlantic salmon and perhapsall salmonids with the ability to exploit a wide variety of freshwaterand seawater environments.

Part 2: Use of RT-PCR and Northern Analysis to Determine the Expressionof SalmoKCaR Clones #1, #2 and #3 in Various Tissues of Atlantic Salmon:

Background:

SalmoKCaR clones #1, #2 and #3 were originally isolated from a Atlanticsalmon kidney cDNA library. To determine the pattern of tissue specificexpression of these various SalmoKCaR clones, both degenerate (toamplify all Salmo PVCRs species) and SalmoKCaR primers that willspecifically amplify either SalmoKCaR #1 or #2 or #3 were utilized. Asshown in “Materials and Methods” Section below, these primers amplifyDNA products of different sizes that can be distinguished by agarose gelelectrophoresis. PCR on specific cDNA clones confirms that these primerpairs function exclusively on the clones for which they have beendesigned. Note that both the degenerate and SalmoKCaR #3 specificprimers do not span an intron and therefore RNA was treated with DNAseto ensure that there was not amplification of contaminating genomic DNAin the results shown. Primers specific for SalmoKCaR #1 and #2 spanintrons and therefore DNAase treatment is not required to interpretthese results. As a control, the amounts of mRNA added to each RT-PCRreaction was determined by separate amplification of actin using primersdesigned from the published sequence of Atlantic salmon actin (GenbankAccession #AF012125 Salmo salar beta actin mRNA).

Materials and Methods:

Primers:

Degenerate Primers

DSK-F3 and DSK-R4 Primers are Shown in Example 4. SalmoKCaR #1 SpecificPrimers SalmoKCaR #1 nts AS1-F17 5′ - CAA GCA TTA TCA AGA TCA AG - 3′ nt47-66 (SEQ ID NO:16) AS2-R14 5′ - CTC AGA GTG GCC TTG GC - 3′ nt2800-2784 (SEQ ID NO:17)

Product from amplification=2754 nt. The SalmoKCaR #1 primer pairconsists of a forward primer (AS1-F17) spanning the 5′ UTR insertion inSalmoKCaR #2, and a reverse primer (AS2-R14) within the 158 bp deletedfrom SalmoKCaR #3. SalmoKCaR #2 Specific Primers SalmoKCaR #2 ntsAS2-F13 5′ - CAG TTC TCT CTT TAA TGG AC - 3′ nt 109-128 (SEQ ID NO:18)AS2-R14 5′ - CTC AGA GTG GCC TTG GC - 3′ nt 2890-2874 (SEQ ID NO:19)

Product from amplification=2782 nt. The SalmoKCaR #2 primer pair is aforward primer (AS2-F13) in the 5′ UTR insertion in SalmoKCaR #2 clone,and the same reverse primer as SalmoKCaR #1 primer (AS2-R14). SalmoKCaR#3 Specific Primers SalmoKCaR #3 nts AS5-F11 5′ - AGT CTA CAT CAT CCATCA GCC -3′ nt 2700-2720 (SEQ ID NO:20) AS5-R12 5′ - GAT TTT ATT GTC ATTGGA TGC - 3′ nt 3810-3790 (SEQ ID NO:21)

Product from amplification=1111 nt. The SalmoKCaR #3 primer pairconsists of a forward primer (AS5-F11) which spans the 158 bp deletion,and a reverse primer (AS5-R12) located in the 36 bp insertion at the 3′end of the SalmoKCaR #3 clone. Salmon Actin Primers SA-F1 5′ - TGG AAGATG AAA TCG CCG C - 3′ nt 2-20 (SEQ ID NO:22) SA-R2 5′ - GTG GTG GTG AAACTG TAA CCG C - 3′ nt 608-587 (SEQ ID NO:23)

Product from amplification=607 nt. This primer set is used to amplifysalmon actin mRNA that serves as a control to quantify differences inmRNA content.

RNA Blotting Analysis and RT-PCR of Atlantic Salmon and ElasmobranchTissues:

Total RNA was purified with Stat 60 reagent (Teltest B Friendswood,Tex.) DNAse (Introgen, Carlsbad, Calif.) treated and used for RT-PCRafter cDNA production with cDNA Cycle Kit (Invitrogen, Carlsbad,Calif.). The cDNA was amplified (30 cycles of 1 min @ 94° C., 1 min @57° C., 3′ @72° C.) using degenerate primers [forward primer dSK-F3(SKCaR nts 2279-2306) and reverse primer dSK-R4 (SKCaR nts 2904-2934).Aliquots of PCR reactions were subjected to gel electrophoresis andethidium bromide (EtBr) staining or blotted onto Magnagraph membranes(Osmonics, Westboro, Mass.) and probed with a ³²P-atlantic salmongenomic PCR product (653 bp sequence identical to that shown in SEQ IDNO: 3 with added nt sequences, washed (0.1×SSC, 0.1% SDS @ 55° C.) andautoradiographed. Selected amplified PCR products from Atlantic salmontissues were sequenced as described above. The following conditions wereutilized for each of the SalmoKCaR specific primers and correspondingblots:

SalmoKCaR #1 amplification conditions and primer set: PCR: 1 min @ 94°C., 1 min @ 50° C., 3 min @ 72° C., 35 cycles. Amplification productsattached to membrane were probed with full length SalmoKCaR #1 clone andwashed (0.1×SSC, 0.1% SDS @ 55° C.) and autoradiographed for 48 hr.

SalmoKCaR #2 amplification conditions and primer set: PCR: 1 min @ 94°C., 1 min @ 50° C., 3 min @ 72° C., 35 cycles. Amplification productsattached to membrane were probed with full length SalmoKCaR #2 clone andwashed (0.1×SSC, 0.1% SDS @ 55° C.) and autoradiographed for 168 hr.

SalmoKCaR #3 amplification conditions and primer set: PCR: 1 min @ 94°C., 1 min @ 52° C., 3 min @ 72° C., 35 cycles. Amplification productsattached to membrane were probed with full length SalmoKCaR #3 clone andwashed (0.1×SSC, 0.1% SDS @ 55° C.) and autoradiographed for 72 hr.

Results:

Analysis of Atlantic Salmon Tissues from Freshwater vs. Seawater AdaptedFish Using Degenerate Primers:

FIG. 17 shows data obtained from 14 tissues of freshwater or seawateradapted Atlantic salmon using the degenerate primers described above.Samples were obtained from a single representative seawater adaptedsalmon (866 gm and 41 cm in length) from a group of 10 fish of averageweight of 678 gm. Samples from nasal lamellae, urinary bladder,olfactory bulb and pituitary gland were all pooled samples from all 10fish. The samples were from a representative single freshwater adaptedfish (112 gm and 21.5 cm) selected from a group of 10 fish with anaverage weight of 142.8 gm. In contrast, samples from nasal lamellae,urinary bladder, olfactory bulb and pituitary gland were all pooledsamples from all 10 fish. Note that the amplification products fromthese degenerate primers do not distinguish between SalmoKCaR #1, #2 or#3 since their nt sequences in the region amplified by the primers areall identical (lanes 7, 9 and 12 Lower gel—Panels A, B, C and D).Moreover, these degenerate primers also possess the capacity to amplifyadditional PVCRs (if any are present) in salmon tissues that could bedistinct from either SalmoKCaR #1-3. Thus, amplified RT-PCR products arereferred to as PVCR products since use of these degenerate primers donot distinguish between various PVCR species.

Analysis of panels A-D of FIG. 17 shows that the PVCR degenerate primersyield PCR products in various tissues of both seawater and freshwateradapted fish. These various bands are more visible in Southern blots(Panels C, D) of corresponding ethidium bromide gels (Panels A and B)because detection of PVCR amplified products via hybridization of a³²P-PVCR probe is more sensitive as compared to ethidium bromidestaining. Prominent ethidium bromide stained bands are visible inurinary bladder (lane 4), kidney (lane 5) and muscle (lane 14) inseawater adapted fish (Panel A) while either faint or no bands are seenin other tissues. In contrast, ethidium bromide bands are also visiblein nasal lamellae (lane 3), urinary bladder (lane 4) and kidney (lane 5)as well as olfactory bulb (lane 12) in freshwater fish (Panel B). Insummary, these data show differential tissue expression of PVCRs

FIG. 17 shows a RT-PCR analysis of freshwater (Panels B, D and F) andseawater (Panels A, C and E) adapted Atlantic salmon tissues usingeither degenerate PVCR or salmon actin PCR primers. Total RNA from 13(seawater adapted) and 14 (freshwater adapted) tissues of Atlanticsalmon was first treated with DNAase to remove any genomic DNAcontamination then used to synthesize cDNA that was amplified usingdegenerate primers. (Panels A and B): Ethidium bromide stained agarosegel. DNA markers in lane 1 of both Panels A and B were used to indicatesize of amplification products. (Panels C and D) Southern blot of gel inTop Panel using ³²P-labeled Atlantic salmon genomic fragment. (Panels Eand F) Ethidium bromide stained gels of RT-PCR amplification productsusing Atlantic salmon beta actin primers as described above. Thesereactions serve as controls to ensure that samples contain equal amountsof RNA.

Southern blots (Panels C and D) of the corresponding gels shown inPanels A and B reveal that amplified PVCR products are present inadditional tissues not shown by simple ethidium bromide staining asdescribed above. As shown in Panel C, PVCRs are present in tissues ofseawater-adapted salmon including gill (lane 2), nasal lamellae (lane3), urinary bladder (lane 4), kidney (lane 5), stomach (lane 6), pyloriccaeca (lane 7), proximal (lane 8) and distal (lane 9) intestine,pituitary gland (11) and muscle (lane 14). Ovary tissue was not testedin seawater-adapted fish. In contrast, freshwater-adapted salmon possessamplified PVCR products in gill (lane 2), nasal lamellae (lane 3),urinary bladder (lane 4), kidney (lane 5), proximal intestine (lane 8),brain (lane 10), pituitary (lane 11), olfactory bulb (lane 12), liver(lane 13), muscle (lane 14) and ovary (lower lane 3). The intensity ofindividual actin bands shown in Panels E and F performed on identicalaliquots of the RT-PCR reactions serve to quantify any differences inpools of cDNA from the individual RT reactions in each sample. Isolationand subcloning of the ethidium bromide stained bands from olfactorylamellae and urinary bladder show that nucleotide sequences of multiplesubclones from these bands all are identical to the nucleotide sequencepresent in SalmoKCaR clones #1-3.

Close examination of the differences in Panel C (seawater adapted) vs.Panel D (freshwater adapted) reveal differences in the apparentabundance of PVCR mRNA in specific tissues. Apparent increases in tissuePVCR mRNA abundance in seawater-adapted salmon vs. freshwater-adaptedsalmon are present in gill, kidney, stomach, pyloric caeca, distalintestine, and muscle. The increased expression of PVCRs in Atlanticsalmon exposed to seawater is consistent with other data that anincrease in PVCR expression in at least one tissue occurs upon transferof Atlantic salmon from freshwater to seawater. In contrast, theabundance of PVCR mRNA species in olfactory bulb tissue of seawateradapted salmon appears to be reduced as compared to olfactory bulbs offreshwater adapted counterparts (Lane 12 in Panels C vs. D). In othertissues such as nasal lamellae (Lane 3 in Panel C vs. D) there is littleor no apparent change in the steady state PVCR mRNA content. In summary,these data demonstrate tissue specific changes in the steady stateexpression of PVCR mRNA species in seawater adapted vs. freshwateradapted Atlantic salmon. Depending on the tissue, steady state PVCR mRNAcontent is either increased, decreased or remains unchanged whenfreshwater adapted fish are compared to seawater adapted counterparts.Since these analyses shown in FIG. 17 use PVCR degenerate primers, it isnot possible to determine from these experiments whether the alterationsin steady state PVCR mRNA content are the result of changes inindividual SalmoKCaRs #1-3.

RT-PCR Analysis Using Degenerate Primers Shows that Steady State Contentof Kidney PVCRs is Increased by the SuperSmolt™ Process Similar to thatProduced by Transfer of Atlantic Salmon to Seawater.

FIG. 18A shows RT-PCR analysis of a single representative experimentwhere kidney tissue was harvested from Atlantic salmon that had eitherbeen freshwater adapted (lane 1), exposed to 9 weeks of the SuperSmolt™process in freshwater (lane 2) or transferred to seawater and maintainedfor 26 days. FIG. 18B shows RT-PCR analysis of a single representativeexperiment using pyloric caeca from the same fish shown in FIG. 18A.Note the significant increase in amplified PVCR product present inkidney (FIG. 18A) and pyloric caeca (FIG. 18B) for both SuperSmolt™(lanes 2 and 7, respectively) and seawater adapted (lanes 3 and 8,respectively) fish as compared to freshwater (lanes 1 and 6,respectively). The increased expression of PVCRs in these 2 tissues ofAtlantic salmon exposed to the SuperSmolt™ process where this increasedPVCR expression mimics that produced after seawater transfer isconsistent with earlier data that an increase in PVCR expression in atleast one tissue occurs upon either treatment with the SuperSmolt™process or transfer of Atlantic salmon to seawater.

FIG. 18 c shows RT-PCR analysis using the same degenerate primers todetect expression of SalmoKCaR transcripts in various stages of Atlanticsalmon embryo development. Using degenerate (SEQ ID Nos 13 and 14) oractin (SEQ ID No 22 and 23) primers, RNA obtained from samples of wholeAtlantic salmon embryos at various stages of development were analyzedfor expression of SalmoKCaRs using RT-PCR. Ethidium bromide staining ofsamples from dechorionated embryos (Lane 1), 50% hatched (Lane 2), 100%hatched (Lane 3), 2 weeks post hatched (Lane 4) and 4 weeks post hatched(Lane 5) shows that SalmoKCaR transcripts are present in Lanes 1-4.Southern blotting of the same gel (Panel C) confirms expression ofSalmoKCaRs in embryos from very early stages up to 2 weeks afterhatching. No expression of SalmoKCaR was observed in embryos 4 weeksafter hatching. Panel B shows the series of controls where PCRamplification of actin content of each of the 5 samples shows they areapproximately equal.

Northern Blotting of Kidney poly A⁺ RNA with SalmoKCaR #1 Reveals anIncrease in PVCR Expression in Seawater-Adapted vs. Freshwater-AdaptedAtlantic Salmon.

To both confirm the size of SalmoKCaR transcripts and test for changesin SalmoKCaR expression in fish exposed to different salinities, poly A⁺RNA from kidney of either freshwater adapted (FW) or seawater adapted(SW) Atlantic salmon were probed with SalmoKCaR #1. As shown in FIG. 19,kidney RNA contains a 4.2 kb band that corresponds to the 3.9-4.0 kbsizes of SalmoKCaR#1-3 as determined by nucleotide sequence analysis.Because of the high degree of nucleotide identities between SalmoKCaR#1-3, the 4.2 kb band is actually derived from the combination of all 3SalmoKCaR species and any additional PVCR species in salmon kidney dueto crosshybridization of SalmoKCaR #1. However, these data show anincrease in the intensity of the 4.2 kb SalmoKCaR band in SW adaptedfish as compared to their FW adapted counterparts.

FIG. 19 shows a RNA blot containing 5 micrograms of poly A⁺ RNA fromkidney tissue dissected from either freshwater adapted (FW) or seawateradapted (SW) Atlantic salmon probed with full length SalmoKCaR #1 clone.Autoradiogram exposure after 7 days.

Use of RT-PCR with SalmoKCaR #3 Specific Primers Demonstrates thatTissue Specific Alterations in the Steady State Tissue Content ofSalmoKCaR #3 mRNA in Freshwater vs. Seawater Adapted Atlantic Salmon.

To determine whether specific SalmoKCaRs #3 are modulated by exposure todifferent salinities, nucleotide primer sets that allows for thespecific amplification of SalmoKCaR transcripts were designed. FIG. 20shows RT-PCR analysis of freshwater (Panels B, D and F) and seawater(Panels A, C and E) adapted Atlantic salmon tissues using eitherSalmoKCaR #3 specific PCR primers or salmon actin PCR primers. Total RNAfrom 13 (seawater adapted) and 14 (freshwater adapted) tissues ofAtlantic salmon identical to those shown in FIG. 17 were first treatedwith DNAase to remove any genomic DNA contamination, then used tosynthesize cDNA that was amplified using SalmoKCaR #3 primers. All RNAsamples were prepared from a single fish with the exception of olfactorybulb, pituitary, urinary bladder and nasal lamellae that are composed ofRNA from pooled samples of fish. Selected reactions were subjected toprimer amplification using SalmoKCaR#3 specific primers. DNA markers inlane 1 of both Panels A and B were used to indicate size ofamplification products. (Panels C and D) Southern blot of gel in TopPanel using ³²P-labeled Atlantic salmon genomic fragment. (Panels E andF) Ethidium bromide stained gel of RT-PCR amplification products usingAtlantic salmon beta actin primers as described above. These reactionsserve as controls to ensure that samples contain equal amounts of RNA.The specificity of these SalmoKCaR#3 primers is demonstrated in thebottom half of Panels A and B of FIG. 20. The specific SalmoKCaR #3primers only amplify product from SalmoKCaR #3 clone (lane 14) and notSalmoKCaR #1 (lane 8) or SalmoKCaR #2 (lane 1 1). Note that in thetissue sample lanes, ethidium bromide stained bands are present in thekidney of seawater adapted salmon (lane 5 upper gel—Panel A) and onlyvery faintly in urinary bladder of freshwater adapted salmon (lane 4upper gel—Panel B). The corresponding Southern blots of freshwateradapted tissue samples (Panel D) reveal detectable SalmoKCaR #3 productonly in urinary bladder (lane 4) and a small amount in kidney (lane 5).In contrast, in seawater-adapted salmon (Panel C) there are detectableincreases in SalmoKCaR #3 product in both urinary bladder (lane 4) andkidney (lane 5) as well as the presence of SalmoKCaR #3 amplifiedproduct in gill (lane 2), nasal lamellae (lane 3), pyloric caeca (lane7) and muscle (lane 14) of seawater adapted fish.

As described above, the increase in tissue expression of SalmoKCaR #3serves to provide for a possible means to reduce the overall tissuesensitivity to PVCR-mediated sensing via an action where SalmoKCaR #3would act as a dominant negative effector. In contrast to freshwaterwhere the ambient water concentrations of both Ca²⁺ and Mg²⁺ are low andrequire a high degree of sensitivity from SalmoKCaRs to sense changes inconcentration, the concentrations of Ca²⁺ and Mg²⁺ in seawater are 10fold and 50 fold higher and thus may require reduction of the highsensitivity of SalmoKCaRs #1 and #2 by SalmoKCaR #3. It is of interestthat many of these specific tissues exhibiting significant SalmoKCaR #3expression are either exposed directly to the high Ca²⁺ and Mg2+ contentof seawater (gill, nasal lamellae) or experience high Ca²⁺ and Mg²⁺concentrations as the result of the excretion of these divalent cations(urinary bladder, kidney).

Use of RT-PCR with SalmoKCaR #1 Specific Primers Demonstrates TissueSpecific Alterations in the Steady State Tissue Content of SalmoKCaR #1mRNA in Freshwater vs. Seawater Adapted Atlantic Salmon.

FIG. 21 shows RT-PCR analysis of freshwater (Panels B, D and F) andseawater (Panels A, C and E) adapted Atlantic salmon tissues usingeither SalmoKCaR #1 specific PCR primers or salmon actin PCR primers.Total RNA from 13 (seawater adapted) and 14 (freshwater adapted) tissuesof Atlantic salmon identical to those shown in FIGS. 17 and 20 were usedto synthesize cDNA that was amplified using SalmoKCaR #1 primers. AllRNA samples were prepared from a single fish with the exception ofolfactory bulb, pituitary, urinary bladder and nasal lamellae that arecomposed of RNA from pooled samples of fish. As controls to demonstrateprimer specificity, selected reactions were subjected to primeramplification of portions of individual SalmoKCaR clones or water alone(Panels A and B): Ethidium bromide stained agarose gel. DNA markers inlane 1 of both Panels A and B were used to indicate size ofamplification products. (Panels C and D) Southern blot of gel in TopPanel using ³²P-labeled Atlantic salmon genomic fragment. (Panes E andF) Ethidium bromide stained gel of RT-PCR amplification products usingAtlantic salmon beta actin primers as described above. These reactionsserve as controls to ensure that samples contain equal amounts of RNA.As shown in lower halves of Panels A and B of FIG. 21, PCR amplificationwith these primers yields an ethidium bromide staining band (lane5) whenSalmoKCaR #1 clone is used as a template but not either SalmoKCaR #2(lane 6) or SalmoKCaR #3 (lane 7). Southern blotting analysis of thegels shown in Panels A and B reveals that the amplification product ofthe SalmoKCaR #3 is highly positive (lanes 5)—Panels C and D. In thevarious tissue samples, SalmoKCaR #1 product is amplified in selectedtissues including urinary bladder (lane 4) and pyloric caeca (lane 7) inseawater-adapted salmon (Panel C) as compared to urinary bladder (lane4) and kidney (lane 5) in freshwater-adapted salmon (Panel D). The exactnature of the smaller and larger than expected PCR amplificationproducts present in gill (lane 2—Panels C, D) and nasal lamellae (lane3—Panel D) are not known at present. These data show tissue specificexpression of SalmoKCaR #1 in both freshwater and seawater adaptedsalmon.

Use of RT-PCR with SalmoKCaR #2 Specific Primers Demonstrates TissueSpecific Alterations in the Steady State Tissue Content of SalmoKCaR #2mRNA in Freshwater vs. Seawater Adapted Atlantic Salmon.

FIG. 22 shows RT-PCR analysis of freshwater (Panels B, D and F) andseawater (Panels A, C and E) adapted Atlantic salmon tissues usingeither SalmoKCaR #2 specific PCR primers or salmon actin PCR primers.Total RNA from 13 (seawater adapted) and 14 (freshwater adapted) tissuesof Atlantic salmon was used to synthesize cDNA that was amplified usingSalmoKCaR #2 primers. All RNA samples were prepared from a single fishwith the exception of olfactory bulb, pituitary, urinary bladder andnasal lamellae that are composed of RNA from pooled samples of fish. Ascontrols to demonstrate primer specificity, selected reactions weresubjected to primer amplification with samples of portions of individualSalmoKCaR clones or water alone (Panels A and B): Ethidium bromidestained agarose gel. DNA markers in lane 1 Panels A, B, E and F wereused to indicate size of amplification products. (Panels C and D)Southern blot of gel in Top Panel using ³²P-labeled Atlantic salmongenomic fragment. (Panes E and F) Ethidium bromide stained gel of RT-PCRamplification products using Atlantic salmon beta actin primers asdescribed above. These reactions serve as controls to ensure thatsamples contain equal amounts of RNA. FIG. 22 shows data obtained usingSalmoKCaR #2 specific primers and the identical tissue RT and plasmidsamples as shown in FIGS. 17, 20, and 21. Corresponding Southern blotsshown in Panels C and D reveal the presence of SalmoKCaR #2 PCRamplification product in urinary bladder of seawater-adapted salmon(lane 4) as well as urinary bladder (lane 4) and kidney (lane 5) offreshwater-adapted salmon. These data provide evidence of the tissuespecific expression of SalmoKCaR #1 in both freshwater and seawateradapted salmon.

EXAMPLE 7 Survival and Growth of Pre-Adult Anadromous Fish by ModulatingPVCRs

An important feature of current salmon farming is the placement of smoltfrom freshwater hatcheries to ocean netpens. Present day methods usesmolt that have attained a critical size of approximately 70-1 10 gramsbody weight. The methods described herein to modulate one or more PVCRsof the anadromous fish including Atlantic Salmon, can either be utilizedboth to improve the ocean netpen transfer of standard 70-110 grams smoltas well as permit the successful ocean netpen transfer of smaller smoltsweighing, for example, only 15 grams. As shown herein, one utility forthe present invention is its use in conjunction with transferringAtlantic Salmon from freshwater to seawater. For standard 70-110 gramsmolt, application of the invention eliminates the phenomenon known as“smolt window” and permits fish to be maintained and transferred intoocean water at 15° C. or higher. Use of these methods in 15 gram orlarger smolt permits greater utilization of freshwater hatcherycapacities followed by successful seawater transfer to ocean netpens. Inboth cases, fish that undergo the steps described herein feed vigorouslywithin a short interval of time after transfer to ocean netpens and thusexhibit rapid growth rates upon transfer to seawater.

FIG. 23 shows in schematic form the key features of current aquacultureof Atlantic salmon in ocean temperatures present in Europe and Chile.Eggs are hatched in inland freshwater hatcheries and the resulting frygrow into fingerlings and parr. Faster growing parr are able to undergosmoltification and placement in ocean netpens as S0 smolt (70 gram)during year 01. In contrast, slower growing parr are smoltified in year02 and placed in netpens as SI smolt (100 gram). In both S0 and S1transfers to seawater, the presence of cooler ocean and freshwatertemperatures are desired to minimize the stress of osmotic shock tonewly transferred smolt. This is particularly true for S1 smolt sincefreshwater hatcheries are often located at significant distances fromocean netpen growout sites and their water temperatures rise rapidlyduring early summer. Thus, the combination of rising water temperaturesand the tendency of smolt to revert or die when held for prolongedintervals in freshwater produces a need to transfer smolt into seawaterduring the smolt window.

Standard smolts that are newly placed in ocean netpens are not able togrow optimally during their first 40-60 day interval in seawater becauseof the presence of osmotic stress that delays their feeding. Thisinterval of osmotic adaptation prevents the smolts from taking advantageof the large number of degree days present immediately after eitherspring or fall placement. The combination of the presence of the smoltwindow together with delays in achieving optimal smolt growth prolongthe growout interval to obtain market size fish. This is particularlyproblematic for S0's since the timing of their harvest is sometimescomplicated by the occurrence of grilsing in maturing fish that areexposed to reductions in ambient photoperiod.

Methods

The smolt were subjected to the steps of Process I and II, as describedherein.

Results and Discussion:

Section I: Demonstration of the Benefits of the Process I For AtlanticSalmon

Demonstration of the Benefits of the Process I For Atlantic Salmon:

Process I increases the survival of small Atlantic Salmon S2 like smoltafter their transfer to seawater when compared to matched freshwatercontrols. Optimal survival is achieved by using the complete processconsisting of both the magnesium and calcium water mixture as well asNaCl diet. In contrast, administration of calcium and magnesium eithervia the food only or without NaCl dietary supplementation does notproduce results equivalent to Process I.

Table 3 shows data obtained from Atlantic salmon S2 like smolts lessthan 1 year old weighing approximately 25 gm. This single group of fishwas apportioned into 4 specific groups as indicated below and each weremaintained under identical laboratory conditions except for thevariables tested. All fish were maintained at a water temperature of9-13° C. and a continuous photoperiod for the duration of theexperiment. The control freshwater group that remained in freshwater forthe initial 45 day interval experienced a 33% mortality rate under theseconditions such that only 67% were able to be transferred to seawater.After transfer to seawater, this group also experienced high mortalitywhere only one half of these smolts survived. Inclusion of calcium (10mM) and magnesium (5 mM) within the feed offered to smolt (Ca2+/Mg2+diet) reduced survival as compared to controls both in freshwater (51%vs 67%) as well after seawater transfer (1% vs 50%). In contrast,inclusion of 10 mM Ca2+ and 5 mM Mg2+ in the freshwater (Process I WaterOnly) improved smolt survival in Process I water as well as aftertransfer of smolt to seawater. However, optimal results were obtained(99% survival in both the Process I water mixture as well as afterseawater transfer) when smolt were maintained in Process I water mixtureand fed a diet supplemented with 7% sodium chloride. TABLE 3 Comparisonof the Survival of Atlantic Salmon S2 like Smolts After VariousTreatments Control Process I Parameter Fresh- Ca2+/Mg2+ Process IWater + Sampled water Diet Water Only NaCl Diet Starting 66 70 74 130 #of fish # of fish 44 36 67 129 % of fish 67%  51%  91%  99%  survivingafter 45 days in freshwater or Process I mixture # of fish 22 2 60 128 %of fish 50%  6% 90%  99%  surviving 5 days after transfer to seawater¹Survival percentages expressed as rounded whole numbersApplication of the Process I to the Placement of 70-100 gm Smolts inSeawater.

These data show that use of the Process I eliminates the “smolt window”and provides for immediate smolt feeding and significant improvement insmolt growth rates.

Experimental Protocol:

Smolts derived from the St. John strain of Atlantic salmon produced bythe Connors Brothers Deblois Hatchery located in Cherryfield, Me., USAwere utilized for this large scale test. Smolts were produced usingstandard practices at this hatchery and were derived from a January 1999egg hatching. All smolts were transferred with standard commerciallyavailable smolt trucks and transfer personnel. SI smolt were purchasedduring Maine's year 2000 smolt window and smolt deliveries were takenbetween the dates of 29 Apr. 2000-15 May 2000. Smolts were eithertransferred directly to Polar Circle netpens (24 m diameter) located inBlue Hill Bay Maine (Controls) or delivered to the treatment facilitywhere they were treated with Process I for a total of 45 days. Afterreceiving the Process I treatment, the smolt were then transported tothe identical Blue Hill Bay netpen site and placed in an adjacentrectangular steel cage (15 m×15 m×5 m) for growout. Both groups of fishreceived an identical mixture of moist (38% moisture) and dry (10%moisture) salmonid feed (Connors Bros). Each of the netpens were fed byhand or feed blower to satiation twice per day using cameravisualization of feeding. Mort dives were performed on a regular basisand each netpen received identical standard care practices establishedon this salmon farm. Sampling of fish for growth analyses was performedat either 42 days (Process I) or 120 days or greater (Control) fish. Inboth cases, fish were removed from the netpens and multiple analysesperformed as described below.

All calculations to obtain feed conversion ratio (FCR) or specificgrowth rate (SGR) and growth factor (GF3) were performed using standardaccepted formulae (Willoughby, S. Manual of Salmonid Farming BlackwellScientific, Oxford UK 1999) and established measurements of degree daysfor the Blue Hill Bay site as provided in Table 4 below. A degree day iscalculated by multiplying the number of days in a month by the meandaily temperature in degrees Celsius. TABLE 4 Degree days for Blue HillBay Salmon Aquaculture Site Month Degree Days January 60 February 30March 15 April 120 May 210 June 300 July 390 August 450 September 420October 360 November 240 December 180

Table 5 displays data obtained after seawater transfer of Control S1smolt. Smolt ranging from 75-125 gm were placed into 3 independentnetpens and subjected to normal farm practices and demonstratedcharacteristics typical for present day salmon aquaculture in Maine.Significant mortalities (average 3.3%) were experienced after transferinto cool (10° C.) seawater and full feeding was achieved only after asignificant interval (˜56 days) in ocean netpens. As a result, theaverage SGR and GF3 values for these 3 netpens were 1.09 and 1.76respectively for the 105-121 day interval measured.

In contrast to the immediate transfer of Control S1 smolt as describedabove to ocean netpens (Table 5), a total of 10,600 S1 smolt possessingan average size of 63.6 grams were transported on 11 May 2000 from theDeblois freshwater hatchery to the research facility. While beingmaintained in standard circular tanks, these fish were held for a totalof 45 days at an average water temperature of 11° C. and were subjectedto Process I. During this interval, smolt mortality was only 64 fish(0.6%). As a matched control for the Process I fish, a smaller group ofcontrol fish (n=220) were held under identical conditions but did notreceive the Process I treatment. The mortalities of these control fishwere minimized by the holding temperature of 10° C. and were equivalentto treated smolts prior to transfer to seawater. TABLE 5 Characteristicsof St. John S1 smolt subjected to immediate placement in ocean netpensafter transport form the freshwater hatchery without Process I orProcess II technology (the Control fish) Netpen Number #17 #18 #10 TotalFish 51,363 43,644 55,570 Mean Date of May 1, 2000 May 5, 2000 May 14,2000 Seawater Transfer Average Size at (117.6) 75-100 75-100 Transfer(grams) 100-125 Mortalities after 30 1,785; 3.5% 728; 1.7% 2503; 4.5%days (# and % total) Time to achieve full 68 days 48 days 50 daysfeeding after transfer Interval between 121 120 105 netpen placement andanalysis Average size at Analysis Weight (gram) 376.8 ± 74 305.80 ± 64298.90 ± 37.40 Length (cm)   33.4 ± 1.9   28.30 ± 9.0 30.40 ± 1.17Condition Factor (k) 1.02 1.34 1.06 SGR 0.96 1.10 1.17 during initial120 days

During the 45 day interval when S1 smolts were receiving Process I, fishgrew an average of 10 grams and thus possessed an average weight of 76.6gm when transferred to an ocean netpen. The actual smolt transfer toseawater occurring on 26 Jun. 2000 was notable for the unusual vigor ofthe smolt that would have normally been problematic since this time iswell past the normal window for ocean placement of smolt. The oceantemperature at the time of Process I smolt netpen placement was 15.1° C.In contrast to the counterpart S1 smolts subjected to standard industrypractices described above, Process I smolts fed vigorously within 48hours of ocean placement and continued to increase their consumption offood during the immediate post-transfer period. The mortality of ProcessI smolts was comparable to that of smolts placed earlier in the summer(6.1%) during initial 50 days after ocean netpen placement and twothirds of those mortalities were directly attributable to scale loss andother physical damage incurred during the transfer process itself.

In contrast, corresponding control fish (held under identical conditionswithout Process I treatment) did not fare well during transfer to thenetpen (17% transfer mortality) and did not feed vigorously at any timeduring the first 20 days after ocean netpen placement. This smallernumber of control fish (176) were held in a smaller (1.5 m×1.5 m×1.5 m)netpen floating within the larger netpen containing Process I smolts.Their mortality post-ocean netpen placement was very high at 63% withinthe 51 day interval.

Both Process I and control smolts were fed on a daily basis in a manneridentical to that experienced by the Industry Standard Fish shown onTable 5. Process I fish were sampled 51 days after their seawaterplacement and compared to the Industry Standard smolts from Table 5. Asshown in Table 6, comparison of their characteristics reveals dramaticdifferences between Industry Standard smolts vs Process I. TABLE 6Comparison of the characteristics of St. John S1 Process I Smoltssubjected to Process I treatment and then placed in ocean netpens vscorresponding industry standard smolts. Averaged Industry Process IStandard Data from Smolts TABLE 5 in this Example Total Fish 10,600150,577 Mean Date of Seawater Jun. 26, 2000 May 7, 2000 Transfer AverageSize at Transfer 76.6 95.8 (grams) Mortalities after 30 days 648; 6.1%5,016; 3.3% (# and %) Time to achieve full 2 days 56 days Feeding aftertransfer Interval between netpen 51 115 placement and analysis Averagesize at Analysis Weight (gram) 175.48 ± 50  327.2 ± 97 Length (cm)  26.2± 32  30.7 Condition Factor (k)  0.95 ± 0.9 1.14 SGR 1.80 1.09

In summary, notable differences between Process I, Control smolt andIndustry Standard smolt include:

1. The mortalities observed after ocean netpen placement were low inProcess I (6.1%) vs Control (63%) despite the that fact these fish weretransferred to seawater 1.5 months after the smolt window and into avery high (15.1° C.) ocean water temperature. The mortality of Process Iwas comparable to that of the accepted Industry Standard smolt (3-10%)transferred to cooler (10C) seawater during the smolt window. Thischaracteristic of Process I provides for a greater flexibility infreshwater hatchery operations since placement of Process I smolts arenot rigidly confined the conventional “smolt window” currently used inindustry practice.

2. The Process I fish were in peak condition during and immediatelyafter seawater transfer. Unlike industry standard smolt that required 56days to reach full feeding, the Process I smolts fed vigorously within 2days. Moreover, the initial growth rate (SGR 1.8) demonstrated byProcess I smolts are significantly greater than published data forstandard smolt during their initial 50 days after seawater placement(published values (Stradmeyer, L. Is feeding nonstarters a waste oftime. Fish Farmer 3:12-13, 1991; Usher, M L, C Talbot and F B Eddy.Effects of transfer to seawater on growth and feeding in Atlantic salmonsmolts (Salmo salar L.) Aquaculture 94:309-326, 1991) for SGR's rangebetween 0.2-0.8). In fact, the growth rates of Process I smolts aresignificantly larger as compared to Industry standard smolts placed intoseawater on the same site despite that industry standard smolt were bothlarger at the time of seawater placement as well as that their growthwas measured 120 days after seawater placement. These data provideevidence that the Process I smolts were not subjected to significantosmoregulatory stress which would prevent them from feeding immediately.

3. The rapid growth of Process I smolts immediately upon ocean netpenplacement provides for compounding increases in the size of salmon asseawater growout proceeds. Thus, it is anticipated that if IndustryStandard Smolts weighing 112.5 gram (gm) were subjected to Process Itreatment, placed in ocean netpens and examined at 120 days after oceannetpen placement their size would be average 782 gram instead of 377gram as observed. This provides for more than a doubling in size of fishin the early stages of growout. Such fish would reach market size morerapidly as compared to industry standard fish.

In contrast to the counterpart SI smolts subjected to standard industrypractices, smolt treated with Process I fed vigorously within 48 hoursof ocean placement and continued to increase their consumption of foodduring the immediate post-transfer period. By comparison, the industrystandard smolts consumed little or no feed within the first week aftertransfer. FIG. 24A compares the weekly feed consumption on a per fishbasis between Process I treated smolts and industry standard smolts. Asshown, Process I treated smolts consumed approximately twice as muchfeed per fish during their FIRST WEEK as compared to the industrystandard smolts after 30 days. Since smolts treated with Process I fedsignificantly more as compared to Industry standard smolts, the ProcessI treated smolts grew faster.

FIG. 24B provides data on the characteristics of Process I smolts afterseawater transfer. These experiments were carried out for over 185 days.

Application of the Process I to Atlantic Salmon Pre-Adult Fish that areSmaller than the Industry Standard “Critical Size” Smolt.

A total of 1,400 Landcatch/St John strain fingerlings possessing anaverage weight of 20.5 gram were purchased from Atlantic Salmon of MaineInc., Quossic Hatchery, Quossic, Me., USA on 1 Aug. 2000. Thesefingerlings were derived from an egg hatching in January 2000 andconsidered rapidly growing fish. They were transported to the treatmentfacility using standard conventional truck transport. After theirarrival, these fingerlings were first placed in typical freshwatergrowout conditions for 14 days. These fingerlings were then subjected toProcess I for a total of 29 days while being exposed to a continuousphotoperiod. The Process I were then vaccinated with the Lipogen Forteproduct (Aquahealth LTD.) and transported to ocean netpens byconventional truck transport and placed into seawater (15.6° C.) ineither a research ocean netpen possessing both a predator net as well asnet openings small enough (0.25 inch) to prevent loss of these smallerProcess I smolts. Alternatively, Process I smolts were placed incircular tanks within the laboratory. Forty eight hours after sea watertransfer, Process I smolts were begun on standard moist (38% moisture)smolt feed (Connors Bros.) that had been re-pelletized due to thenecessity to provide for smaller size feed for smaller Process I smolts,as compared to normal industry salmon. In a manner identical to thatdescribed for 70 gram smolts above, the mortality, feed consumption,growth and overall health of these 30 gram Process I smolts weremonitored closely.

FIG. 25 displays the characteristics of a representative sample of alarger group of 1,209 Process I smolts immediately prior to theirtransfer to seawater. These parameters included an average weight of26.6+8.6 gram, length of 13.1+1.54 cm and condition factor of 1.12+0.06.After seawater transfer, Process I smolts exhibited a low initialmortality despite the fact that their average body weight is 26-38% ofindustry standard 70-100 gram SO-SI smolts. As shown in Table 7, ProcessI smolts mortality within the initial 72 hr after seawater placement was1/140 or 0.7% for the laboratory tank. Ocean netpen mortalities afterplacement of Process I smolts were 143/1069 or 13.4%. FIG. 25 showsrepresentative Landcatch/St John strain Process I smolts possessing arange of body sizes that were transferred to seawater either in oceannetpens or corresponding laboratory seawater tanks. Process I smoltspossess a wide range of sizes (e.g., from about 5.6 grams to about 46.8grams body weight) with an average body weight of 26.6 gram. Experimentswith these data were carried out for 84 days after the transfer of fishto seawater tanks, and the data from these experiments are described inco-pending application Ser. No. 09/975,553, Attorney Docket No:2213.1004-001. TABLE 7 Characteristics and survival of Landcatch/St.John Process I fish after their placement into seawater in either alaboratory tank or ocean netpen. Laboratory Tank Ocean Netpen Total Fish140 1,069 Date of Seawater Transfer  Sep. 5, 2000 (40); Sep. 12, 2000Sep. 12, 2000 (100) Average Size at Transfer 26.6 26.6 (gram) Totalmortalities after 4 1; 0.7% 143; 13.4% days (# and % total) % mortalityof fish 0; 0.0%  4; 0.4% weighing 25 gm and above Time to achievefeeding 48 hrs 72 hrs

FIG. 26 shows a comparison of the distributions of body characteristicsfor total group of Landcatch/St John Process I smolts vs. mortalities 72hr after seawater ocean netpen placement. Length and body weight dataobtained from the 143 mortalities occurring after seawater placement of1,069 Process I smolts were plotted on data obtained from a 100 fishsampling as shown previously in FIG. 25. Note that the mortalities areexclusively distributed among the smaller fish within the larger ProcessI netpen population.

Length and weight measurements for all mortalities collected from thebottom of the ocean netpen were compared to the distribution of ProcessI smolt body characteristics obtained from analysis of a representativesample prior shown in FIG. 26. The data show that the mortalitiesoccurred selectively amongst Process I smolts possessing small bodysizes such that the mean body weight of mortalities was 54% of the meanbody weight of the total transfer population (14.7/27 gram or 54%).Thus, the actual mortality rates of Process I smolts weighing 25-30 gramis 0.4% (4/1069) and those weighing 18-30 gram is 2.9% (31/1069).

Application of Process I to Trout Pre-Adult Fish that are Smaller thanthe Industry Standard “Critical Size” Smolt.

Table 8 displays data on the use of the Process I on small (3-5 gram)rainbow trout. Juvenile trout are much less tolerant of abrupt transfersfrom freshwater to seawater as compared to juvenile Atlantic salmon. Asa result, many commercial seawater trout producers transfer their fishto brackish water sites located in estuaries or fresh water lenses orconstruct “drinking water” systems to provide fresh water for troutinstead of the full strength seawater present in standard ocean netpens.After a prolonged interval of osmotic adaptation, trout are thentransferred to more standard ocean netpen sites to complete theirgrowout cycle. In general, trout are transferred to these ocean sitesfor growout at body weights of approximately 70-90 or 90-120 gram. TABLE8 Comparison of the Survival of Rainbow Trout (3-5 gram) in SeawaterAfter Various Treatments. Percent Survival of Fish¹ Constant 14 Constant23 Hours Post Control Constant 14 day day Seawater Fresh- dayPhotoperiod Photoperiod + Transfer water Photoperiod Process I Process I0 100 100 100 100 24 0 25 80 99 48 0 70 81 72 40 68 96 30 58 120 30 46Number of 10 20 30 80 Fish Per Experiment¹Survival percentages expressed as rounded whole numbers

A total of 140 trout from a single pool of fish less than 1 year oldwere divided into groups and maintained at a water temperature of 9-13°C. and pH 7.8-8.3 for the duration of the experiment described below.When control freshwater rainbow trout are transferred directly intoseawater, there is 100% mortality within 24 hr (Control Freshwater).Exposure of the trout to a constant photoperiod for 14 days results in aslight improvement in survival after their transfer to seawater. Incontrast, exposure of trout to Process I for either 14 days or 23 daysresults in significant reductions in mortalties after transfer toseawater such that 30% and 46% of the fish respectively have survivedafter a 5 day interval in seawater. These data demonstrate thatapplication of the Process I increases in the survival of pre-adulttrout that are less than 7% of the size of standard “critical size”trout produced by present day industry standard techniques.

Application of the Process I to Arctic Char Pre-Adult Fish that areSmaller than the Industry Standard “Critical Size” Smolt.

Although arctic char are salmonids and anadromous fish, their toleranceto seawater transfer is far less as compared to either salmon or trout.FIG. 27 shows the results of exposure of smaller char (3-5 gram) to theProcess I for a total of 14 and 30 days. All fish shown in FIG. 27 wereexposed to a continuous photoperiod. Transfer of char to seawaterdirectly from freshwater results in the death of all fish within 24 hr.In contrast, treatment of char with the Process I for 14 and 30 daysproduces an increase in survival such that 33% (3/9) or 73% (22/30)respectively are still alive after a 3 day exposure. These datademonstrate that the enhancement of survival of arctic char that areless than 10% of the critical size as defined by industry standardmethods after their exposure to the Process I followed by transfer toseawater.

FIG. 27 shows a comparison of survival of arctic char after varioustreatments. A single group of arctic char (3-5 gram were obtained fromPierce hatcheries (Buxton, Me.) and either maintained in freshwater ortreated with the Process I prior to transfer to seawater.

Section II: The Use of the Process II to Permit Successful Transfer of10-30 Gram Smolt into Seawater Netpens and Tanks.

The Process II protocol is utilized to treat pre-adult anadromous fishfor placement into seawater at an average size of 25-30 gram or less.This method differs from the Process I protocol by the inclusion ofL-tryptophan in the diet of pre-adult anadromous fish prior to theirtransfer to seawater. Process II further improves the osmoregulatorycapabilities of pre-adult anadromous fish and provides for still furtherreductions in the “critical size” for Atlantic salmon smolt transfers.In summary, Process II reduces the “critical size” for successfulseawater transfer to less than one fifth the size of the present dayindustry standard SO smolt.

Application of Process II to Atlantic Salmon Fingerlings:

St John/St John strain pre-adult fingerlings derived from a January 2000egg hatching and possessing an average weight of 0.8 gram were purchasedfrom Atlantic Salmon of Maine Inc. Kennebec Hatchery, Kennebec Me. on 27Apr. 2000. These fish were transported to the treatment facility usingstandard conventional truck transport. After their arrival, these parrwere first grown in conventional flow through freshwater growoutconditions that included a water temperature of 9.6° C. and a standardfreshwater parr diet (Moore-Clark Feeds). On 17 Jul. 2000, fingerlingswere begun on Process II for a total of 49 days while being exposed to acontinuous photoperiod. Process II smolts were then vaccinated with theLipogen Forte product (Aquahealth LTD.) on Day 28 (14 Aug. 2000) ofProcess II treatment. Process II smolts were size graded prior toinitiating Process II as well as immediately prior to transfer toseawater. St John/St John Process II smolts were transported to oceannetpens by conventional truck transport and placed into seawater (15.2°C.) in either a single ocean netpen identical to that described forplacement of Process I smolts or into laboratory tanks (15.6° C.) withinthe research facility.

FIG. 28 shows representative St. John/St John strain Process II smoltspossessing a range of body sizes were transferred to seawater either inocean netpens or corresponding laboratory seawater tanks. Note thatthese Process II smolts possess a wide range of body weights (3.95-28gram) that comprised an average body weight of 11.5 gram. FIG. 28 showsthe characteristics of St. John/St John Process II smolts. The averagemeasurements of these St. John/St. John Process II smolts included abody weight of 11.50±5.6 gram, length of 9.6±1.5 cm and condition factorof 1.19±0.09. The data displayed in Tables 9 and 10 show the outcomesfor two groups of Process II smolts derived from a single productionpool of fish after their seawater transfer into either laboratory tanksor ocean netpens. Although important variables such as the watertemperatures and transportation of fish to the site of seawater transferwere identical, these 2 groups of Process II smolts experienceddifferential post seawater transfer mortalities after 5 days intolaboratory tanks (10% mortality) and ocean netpens (37.7% mortality).

The probable explanation for this discrepancy in mortalities betweenseawater laboratory tanks (10% mortality) and ocean netpens (37.7%mortality) is exposure of these fish to different photoperiod regimensafter seawater placement. Exposure of juvenile Atlantic salmon to aconstant photoperiod after seawater placement reduced theirpost-seawater transfer mortality from approximately 34% to 6%. Fishtransferred to ocean netpens experienced natural photoperiod that wasnot continuous and thus suffered an approximate 4-fold increase inmortality. As shown in Table 9, a separate seawater transfer of StJohn/St John juvenile Atlantic salmon possessing an average weight of 21gms exhibited only 0.2% mortality after a six week treatment withProcess II and underwater lights. These fish were exposed to acontinuous photoperiod by underwater halogen lights for an interval of30 days. TABLE 9 Characterization and survival of St. John/St. JohnProcess II fish after their placement into seawater in ocean netpenscontaining underwater lights. Total Fish 15,000 Seawater Transfer DateAug. 9, 2001 Water Temperature (oC) 12.6 Size at Transfer (gram)21+/−4.5 Total Mortalities after 30 days (# and % total) 250 1.7% %Mortalities weighing 15 grams or greater 30 0.2% Time to achieve feedingafter transfer 48 hr

TABLE 10 Characteristics and survival of St. John/St. John Process IIfish after their placement into seawater in either a laboratory tank orocean netpen. Laboratory Tank Ocean Netpen Total Fish 100 1,316 SeawaterTransfer Date Aug. 31, 2000 Sep. 5, 2000 Water Temperature (° C.) 15.615.6 Size at Transfer (gram) 11.5 11.5 Total Mortalities after 5 10; 10%496; 37.7% days (# and % total) % mortalities weighing 13 0; 0% 1; 0.08%grams or greater Time to achieve feeding 48 hrs 48 hrs after transfer

No apparent problems were observed with the smaller (10-30 gram) ProcessII smolts negotiating the conditions that exist within the confines oftheir ocean netpen. This included the lack of apparent problemsincluding the ability to school freely as well as the ability to swimnormally against the significant ocean currents that are continuouslypresent in the commercial Blue Hill Bay salmon aquaculture site. Whilethese observations are still ongoing, these data do not suggest that theplacement and subsequent growth of Process II smolts in ocean netpenswill be comprised because of lack of ability of these pre-adultanadromous fish to swim against existing ocean currents and therefore beunable to feed or develop properly.

FIG. 29 compares characteristics of survivors and mortalities of ProcessII smolts after seawater transfer to either laboratory tanks (FIG. 29A)or ocean netpens (FIG. 29B). FIG. 29A data are derived from analyses of100 Process II smolts transferred to seawater tank where all fish werekilled and analyzed on Day 5. In contrast, FIG. 29B displays onlymortality data from ocean netpen. In both cases, only smaller Process IIsmolts experienced mortality. Note differences in Y axis scales of FIGS.29A-B.

Comparison of the average body size of those Process II smolts thatsurvived seawater transfer vs. those Process II smolts that died showsthat unsuccessful Process II smolts possessed significantly smaller bodyweights as compared to average body size of whole Process II smolttransfer group. Thus, the average weight of mortalities in laboratorytank (5.10±2.2 gram) and ocean netpen (6.46±1.5 gram) are 44% and 56%respectively the value of the average body weight possessed by theentire transfer cohort (11.5 gram). In contrast, the mortalities ofProcess II smolts with body weights greater than 13 gram is 0/100 in thelaboratory tank and 1/1316 or 0.076% for ocean netpens. Together, thesedata demonstrate that Process II is able to redefine the “critical size”of Atlantic salmon smolts from 70-100 gram to approximately 13 gram.

Quantitation of Feeding and Growth of Process I and II Smolts afterSeawater Transfer:

Landcatch/St John Process I smolts were offered food beginning 48 hrafter their seawater transfer to either laboratory tanks or oceannetpens. While these Process I smolts that were transferred tolaboratory tanks began to feed after 48 hr, those fish transferred toocean netpens were not observed to feed substantially until 7 days. Tovalidate these observations, the inventors performed direct visualinspection of the gut contents from a representative sample of 49Process I smolts 4 days after their seawater transfer to laboratorytanks. A total of 21/49 or 42.9% possessed food within their gutcontents at that time.

The St John/St John Process II smolts fed vigorously when first offeredfood 48 hrs after their seawater transfer regardless of whether theywere housed in laboratory tanks or ocean netpens. An identical directanalysis of Process II smolts gut contents performed as described aboverevealed that 61/83 or 73.5% of fish were feeding 4 days after transferto seawater. The vigorous feeding activity of Process II smolts in anocean netpen as well as laboratory tanks occurred. Taken together, thesedata suggest that Process I and II smolts do not suffer from a prolonged(20-40 day) interval of poor feeding after seawater transfer as isnotable for the much larger industry standard Atlantic salmon smolts nottreated with the process. The growth rates of identical fish treatedwith either Process I or II within laboratory seawater tanks has beenquantified. As shown in Table 11, both Atlantic salmon treated withProcess I or II grow rapidly during the initial interval (21 days) aftertransfer to seawater. In contrast to industry standard smolt weighing70-100 grams that eat poorly and thus have little or no growth duringtheir first 20-30 days after transfer to seawater, pre-adult Atlanticsalmon receiving Process I or II both exhibited substantial weight gainsand growth despite the fact that they are only 27-38% (Process I) and12-16% (Process II) of the critical size of industry standard smolts.Data that relates to mortalities, SGR, temperature corrected SGR (GF3),FCR, body weights, lengths and condition factors for these same fishwere obtained a total of 4 additional intervals during an interval thatnow extends for 157 days. TABLE 11 Comparison of Growth Rates ofPre-adult Atlantic Salmon Exposed to either Process I or Process II andPlaced in Laboratory Tanks During Initial Interval After SeawaterTransfer Process I Process II Number of Fish 140 437 Weight at Placement26.6 11.50 into Seawater Days in Seawater 22 21 Placement Weight 26.6*13.15* Corrected for Mortalities Weight after Interval 30.3 15.2 inSeawater Weight Gained in 3.75 2.05 Seawater SGR (% body 0.60 0.68weight/day) FCR 1.27 2.04*Weight gain corrected for selective mortalities amongst smaller fish(4/140 or 2.9% Process I; 103/437 or 23.6% Process II)

EXAMPLE 8 Exposure of Salmon Smolts TO Ca2+ and Mg2+ IncreasesExpression of PVCR in Certain Tissues

In smolts that were exposed to 10 mM Ca²⁺ and 5.2 mM Mg²⁺, theexpression of PVCR was found to increase in a manner similar to that insmolts that are untreated, but are transferred directly to seawater.

Tissues were taken from either Atlantic salmon or rainbow trout, afteranesthesitizing the animal with MS-222. Samples of tissues were thenobtained by dissection, fixed by immersion in 3% paraformaldehyde,washing in Ringers then frozen in an embedding compound, e.g., O.C.T.™(Miles, Inc., Elkahart, Ind., USA) using methylbutane cooled on dry ice.After cutting 8 micron thick tissue sections with a cryostat, individualsections were subjected to various staining protocols. Briefly, sectionsmounted on glass slides were: 1) blocked with goat serum or serumobtained from the same species of fish, 2) incubated with rabbitanti-CaR antiserum, and 3) washed and incubated withperoxidase-conjugated affinity-purified goat antirabbit antiserum. Thelocations of the bound peroxidase-conjugated goat anti-rabbit antiserumwere visualized by development of a rose-colored aminoethylcarbazolereaction product. Individual sections were mounted, viewed andphotographed by standard light microscopy techniques. The methods usedto produce anti-PVCR antiserum are described below.

The results are shown in FIGS. 30A-30G, which are a set of sevenphotomicrographs showing immunocytochemistry of epithelia of theproximal intestine of Atlantic salmon smolts using anti-PVCR antiserum,and in FIG. 31, which is a Western blot of intestine of a salmon smoltexposed to Ca2+- and Mg2+-treated freshwater, then transferred toseawater. The antiserum was prepared by immunization of rabbits with a16-mer peptide containing the protein sequence encoded by the carboxylterminal domain of the dogfish shark PVCR (“SKCaR”) (Nearing, J. et al.,1997, J. Am. Soc. Nephrol. 8:40A). Specific binding of the anti-PVCRantibody is indicated by aminoethylcarbazole (AEC) reaction product.

FIGS. 30A and 30B show stained intestinal epithelia from smolts thatwere maintained in freshwater then transferred to seawater and held foran interval of 3 days. Abundant PVCR immunostaining is apparent in cellsthat line the luminal surface of the intestine. The higher magnification(1440×) shown in FIG. 30B displays PVCR protein localized to the apical(luminal-facing) membrane of intestinal epithelial cells. The pattern ofPVCR staining is localized to the apical membrane of epithelial cells(small arrowheads) as well as membranes in globular round cells(arrows). FIG. 30C shows stained intestinal epithelia from arepresentative smolt that was exposed Process I and maintained infreshwater containing 10 mM Ca2+ and 5.2 mM Mg2+ for 50 days. Note thatthe pattern of PVCR staining resembles the pattern exhibited byepithelial cells displayed in FIGS. 30A and 30B including apicalmembrane staining (small arrowheads) as well as larger globular roundcells (arrows). FIG. 30D shows a 1900× magnification of PVCR-stainedintestinal epithelia from another representative fish that was exposedto the Process I and maintained in freshwater containing 10 mM Ca2+ and5.2 mM Mg2+ for 50 days and fed 1% NaCl in the diet. Again, smallarrowhead and arrows denote PVCR staining of the apical membrane andglobular cells respectively. In contrast to the prominent PVCR stainingshown in FIGS. 30A-D, FIGS. 30E (1440×) and 13F (1900×) show staining ofintestinal epithelia from two representative smolt that were maintainedin freshwater alone without supplementation of Ca2+ and Mg2+ or dietaryNaCl. Both 13E and 13F display a marked lack of significant PVCRstaining. FIG. 30G (1440×) shows the lack of any apparent PVCR stainingupon the substitution of preimmune serum on a section corresponding tothat shown in FIG. 30A where anti-PVCR antiserum identified the PVCRprotein. The lack of any PVCR staining with preimmune antiserum is acontrol to demonstrate the specificity of the anti-PVCR antiserum underthese immunocytochemistry conditions. The relative amount of PVCRprotein present in intestinal epithelial cells of freshwater smolts(FIGS. 30E and 30F) was negligible as shown by the faint staining ofselected intestinal epithelial cells. In contrast, the PVCR proteincontent of the corresponding intestinal epithelial cells wassignificantly increased upon the transfer of these smolts to seawater(FIGS. 30A and 30B). Importantly, the PVCR protein content was alsosignificantly increased in the intestinal epithelial cells of smoltsmaintained in freshwater supplemented with Ca2+ and Mg2+ (FIGS. 30C and30D). The AEC staining was specific for the presence of the anti-PVCRantiserum, since substitution of the immune antiserum by the preimmuneeliminated all reaction product from intestinal epithelial cell sections(FIG. 30G).

Disclosure of Localization of PVCR Protein(s) in Additional Areas ofOsmoregulatory Organs of Atlantic Salmon using Paraffin Sections.Demonstration that PVCR Proteins are Localized to both the Apical andBasolateral Membranes of Intestinal Epithelial Cells.

Using the methods described herein, immunolocalization data fromparaffin sections of various osmoregulatory organs of seawater-adaptedjuvenile Atlantic salmon smolt were obtained. PVCR proteins, asdetermined by the binding of a specific anti-PVCR antibody, were presentin the following organs. These organs are important in variousosmoregulatory functions. These organs include specific kidney tubulesand urinary bladder responsible for processing of urine, and selectedcells of the skin, nasal lamellae and gill each of which are bathed bythe water surrounding the fish. The PVCR was also seen in variousportions of the G.I. tract including stomach, pyloric caeca, proximalintestine and distal intestine that process seawater ingested by fish.These tissues were analyzed after treatment with Processes I and II, andafter their transfer from freshwater to seawater. In addition, it isbelieved that the PVCR protein can also act as a nutrient receptor forvarious amino acids that are reported to be present in stomach, proximalintestine, pyloric caeca.

In particular, higher magnification views of PVCR immunolocalizations inselected cells of the stomach, proximal intestine and pyloric caeca wereobtained. The PVCR protein is not only present on both the apical(luminally facing) and basolateral (blood-facing) membranes of stomachepithelial cells localized at the base of the crypts of the stomach, butalso is present in neuroendocrine cells that are located in thesubmucosal area of the stomach. From its location on neuroendocrinecells of the G.I. tract, the PVCR protein is able to sense the localenvironment immediately adjacent to intestinal epithelial cells andmodulate the secretion and synthesis of important G.I. tract hormones(e.g., 5-hydroxytryptamine (5-HT), serotonin, or cholecystokinin (CCK)).Importantly, it is believed that the constituents of Process II effectG.I. neuroendocrine cells by at least two means. The first way thatconstituents of Process II remodel the G.I endocrine system is throughalterations in the expression and/or sensitivity of PVCRs expressed bythese cells. The second way is to supply large quantities of precursorcompounds, for example, tryptophan that is converted into 5-HT andserotonin by G.I. metabolic enzymes.

In a similar manner, PVCR protein is localized to both the apical andbasolateral membranes of epithelial cells lining the proximal intestine.From their respective locations, PVCR proteins can sense both theluminal and blood contents of divalent cations, NaCl and specific aminoacids and thereby integrate the multiple nutrient and ionabsorptive-secretory functions of the intestinal epithelial cells.Epithelial cells of pyloric caeca also possess abundant apical PVCRprotein.

To further demonstrate the specificity of the anti-CaR antiserum torecognize salmon smolt PVCRs, FIG. 31 shows a Western blot of intestinalprotein from salmon smolt maintained in 10 mM Ca2+, 5 mM Mg2+ and fed 1%NaCl in the diet. Portions of the proximal and distal intestine werehomogenized and dissolved in SDS-containing buffer, subjected toSDS-PAGE using standard techniques, transferred to nitrocellulose, andequal amounts of homogenate proteins as determined by both protein assay(Pierce Chem. Co, Rocford, Ill.) as well as Coomassie Blue staining wereprobed for presence of PVCR using standard western blotting techniques.The results are shown in the left lane, labeled “CaR”, and shows a broadband of about 140-160 kDa and several higher molecular weight complexes.The pattern of PVCR bands is similar to that previously reported forshark kidney (Nearing, J. et al., 1997, J. Am. Soc. Nephrol. 8:40A) andrat kidney inner medullary collecting duct (Sands, J. M. et al., 1997,J. Clin. Invest. 99:1399-1405). The lane on the right was treated withthe preimmune anti-PVCR serum used in FIG. 30G, and shows a completelack of bands. Taken together with immunocytochemistry data shown inFIG. 30, this immunoblot demonstrates that the antiserum used isspecific for detecting the PVCR protein in salmon.

EXAMPLE 9 Immunolocalization of Polyvalent Cation Receptor (PVCR) inMucous Cells of Epidermis of Salmon

The skin surface of salmonids is extremely important as a barrier toprevent water gain or loss depending whether the fish is located infresh or seawater. Thus, the presence of PVCR proteins in selected cellsof the fish's epidermal layer would be able to “sense” the salinity ofthe surrounding water as it flowed past and provide for the opportunityfor continuous remodeling of the salmonid's skin based on thecomposition of the water where it is located.

Methods: Samples of the skin from juvenile Atlantic Salmon resident inseawater for over 12 days were fixed in 3% paraformaldehyde dissolved inbuffer (0.1M NaP04, 0.15M NaCl, 0.3M sucrose pH 7.4), manually descaled,rinsed in buffer and frozen at −80° C. for cryosectioning. Ten micronsections were either utilized for immunolocalization of PVCR usinganti-shark PVCR antiserum or stained directly with 1% Alcian Blue dye tolocalize cells containing acidic glycoprotein components of mucous.

Results and Discussion: FIG. 32A shows that salmon epidermis containsmultiple Alcian Blue staining cells present in the various skin layers.Note that only a portion of some larger cells (that containing acidicmucins) stains with Alcian Blue (denoted by the open arrowheads). Forpurposes of orientation, note that scales have been removed so asterisksdenote surface that was previously bathed in seawater. FIG. 32B showsimmunolocalization of salmon skin PVCR protein that is localized tomultiple cells (indicated by arrowheads) within the epidermal layers ofthe skin. Note that anti-PVCR staining shows the whole cell body, whichis larger than its corresponding apical portion that stains with AlcianBlue as shown in FIG. 32A. The presence of bound anti-CaR antibody wasindicated by the rose color reaction product. Although formalquantitation has not yet been performed on these sections, it appearsthat the number of PVCR cells is less than the total number of AlcianBlue positive cells. These data indicate that only a subset of AlcianBlue positive cells contain abundant PVCR protein. FIG. 32C shows theControl Preimmune section where the primary anti-PVCR antiserum wasomitted from the staining reaction. Note the absence of rose coloredreaction product in the absence of primary antibody.

These data demonstrate the presence of PVCR protein in discreteepithelial cells (probably mucocytes) localized in the epidermis ofjuvenile Atlantic salmon. From this location, the PVCR protein could“sense” the salinity of the surrounding water and modulate mucousproduction via changes in the secretion of mucous or proliferation ofmucous cells within the skin itself. The PVCR agonists (Ca2+, Mg2+)present in the surrounding water activate these epidermal PVCR proteinsduring the interval when smolts are being exposed to the process of thepresent invention. This treatment of Atlantic salmon smolts by theprocess of the present invention is important to increased survival ofsmolts after their transfer to seawater.

EXAMPLE 10 Demonstration of the Use of Solid Phase Enzyme-Linked Assayfor Detection of PVCRs in Various tissues of Individual Atlantic Salmonusing Anti-PVCR Polyclonal Antiserum

The PVCR content of various tissues of fish can be quantified using anELISA 96 well plate assay system. The data, described herein,demonstrate the utility of a 96 well ELISA assay to quantify the tissuecontent of PVCR protein using a rabbit polyclonal anti-PVCR antibodyutilized to perform immunocytochemistry and western blotting. These dataform the basis for development of commercial assay kits that wouldmonitor the expression levels of PVCR proteins in various tissues ofjuvenile anadromous fish undergoing the processes of the presentinvention, as described herein. The sensitivity of this ELISA isdemonstrated by measurement of the relative PVCR content of 14 tissuesfrom a single juvenile Atlantic salmon, as shown in FIG. 33.

Description of Experimental Protocol:

Homogenates were prepared by placing various tissues of juvenileAtlantic salmon (St. John/St. John strain average weight 15-20 gm) intoa buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1 mMPhenylmethylsulfonyl fluoride (PMSF), 0.5 dithiothreitol (DTT) and 1 mMbenzamidine pH 8.8) and using a standard glass Potter-Elvenhiemhomogenizer with a rotary pestle. After centrifugation at 2,550×g for 20min. at 4° C. to remove larger debris, the supernatant was either useddirectly or frozen at −80° C. until further use. Homogenate proteinconcentrations were determined using the BCA assay kit (Pierce Chem.Co.). Aliquots of individual tissue homogenates were diluted into aconstant aliquot size of 100 microliters and each was transferred to a96 well plate (Costar Plastic Plates) and allowed to dry in room air for15 hr. After blocking of nonspecific binding with a solution of 5%nonfat milk powder +0.5% Tween 20 in TBS (25 mM Tris 137 mM sodiumchloride, 2.7 mM KCl pH 8.0), primary antiserum (either rabbit anti-PVCRimmune or corresponding rabbit pre-immune antiserum) at a 1:1500dilution was added. After a I hr incubation, individual wells wererinsed 3 times with 500 microliters of TBS, an 1:3000 horseradishperoxidase conjugated goat anti-rabbit (Gibco-BRL) were added andallowed to incubate for 1 hr. Individual wells were then rinsed andbound complex of primary-secondary antibody detected with Sigma A32192,2′Azino-bis(3-ethylbenzthiazidine-6-sulfonic acid) color reagent after15 min of incubation using a Molecular Devices 96 well plate reader(Molecular Devices, VMAX) at 405 nm. Relative amounts of tissue PVCRcontent were determined after corrections for minimal background andnonspecific antibody binding as measured by binding of preimmuneantiserum.

Results and Data Interpretation:

FIG. 33 shows the data obtained from a representative single ELISAdetermination of PVCR protein content of 14 tissues of a single juvenileAtlantic salmon. Under the conditions specified in the ExperimentalProtocol as outlined above, nonspecific binding of both primary andsecondary antibodies were minimized. While these quantitative values aremeasured relative to each other and not in absolute amounts, theyprovide data that parallels extensive immunocytochemistry examination ofeach of the tissues. Note that the PVCR content of various organsreflects their importance in osmoregulation of Atlantic salmon.Immunocytochemistry data described herein shows that tissues such asintestine (proximal and distal segments), gill, urinary bladder andkidney contained PVCR protein. In each case, epithelial cells thatcontact fluids that bathe the surfaces of these tissues express PVCR. Incontrast, other organs including liver, heart and muscle contain minimalPVCR protein. Note that the highest PVCR content of any tissue tested isthe olfactory lamellae where salmon possess the ability to “smell”alterations in calcium concentration in water. The olfactory bulbcontaining neurons that innervate the olfactory lamellae also possessabundant PVCR. Taken together, these data demonstrate the utility ofELISA kits to measure tissue content of PVCR proteins and form the basisfor development of commercial assay kits that would monitor theexpression levels of PVCR proteins in various tissues of juvenileanadromous fish undergoing the processes of the present invention.Alterations in PVCR tissue content measured in either relative changesin tissue PVCR content or absolute quantity of PVCR per tissue masscould, in turn, be utilized as correlative assays to determine thereadiness of juvenile anadromous fish for sea water transfer orinitiation of feeding. These data demonstrate the ability to performsuch assays on individual juvenile Atlantic salmon in the range of bodysizes that would be utilized to transfer fish from fresh to seawaterafter treatment with the methods of the present invention.

EXAMPLE 11 Antibodies Made from the Carboxyl Terminal Portion of anAtlantic Salmon PVCR Protein are Effective in Immunocytochemistry andImmunoblotting Assays to Determine the Presence, Absence or Amount ofthe PVCR Protein

Degenerate primers, dSK-F3 (SEQ ID NO: 13) and dSK-R4 (SEQ ID NO: 14),described herein were constructed specifically from the SKCaR DNAsequence. These primers have proved to be useful reagents foramplification of portions of PVCR sequences from both genomic DNA aswell as cDNA.

To obtain more cDNA sequence from anadromous fish PVCRs, in particularthe putative amino acid sequence of the carboxyl terminal domain ofPVCRs that are targets for generation of specific peptides and, as aresult, specific anti-Atlantic Salmon PVCR antisera, an unamplified cDNAlibrary from Atlantic salmon intestine was constructed. Phage plaquesoriginating from this cDNA library were screened under high stringencyusing ³²P-labeled 653 bp genomic Atlantic Salmon PCR product. From thiscDNA library screening effort, a 2,021 bp cDNA clone was isolated andcontained a single open reading frame for a putative amino acid sequencecorresponding to approximately one half of a complete cDNA sequence froman intestinal PVCR protein. This putative amino acid sequencecorresponds exactly to the sequence encoded by the corresponding genomicprobe as well as the putative amino acid sequence corresponding to thecarboxyl terminal domain of the PVCR.

On the basis of the knowledge of this putative amino acid sequence, apeptide, shown below, was synthesized and corresponded to a separateregion of the putative carboxyl terminal PVCR amino acid sequence:

The peptide sequence for antibody production is as follows: Peptide #1:Ac-CTNDNDSPSGQQRIHK-amide (SEQ ID NO.:15)producing rabbit antiserum SAL-1

The peptide was derivatized to carrier proteins and utilized to raisepeptide specific antiserum in two rabbits using methods for making apolyclonal antibody.

The resulting peptide specific antiserum was then tested using bothimmunoblotting and immunocytochemistry techniques to determine whetherthe antibody bound to protein bands corresponding to PVCR proteins oryielded staining patterns similar to those produced using otheranti-PVCR antiserum. A photograph of an immunoblot was taken showingprotein bands that were recognized by antisera raised against peptidescontaining either SAL-1 (SEQ ID NO.: 15) or SKCaR (SEQ ID NO:2). Asexpected, antiserum raised to the peptide identified protein bands thatco-electrophorese with PVCR proteins that are recognized by antiseraraised to SKCaR (SEQ ID NO:2). Immunostaining of juvenile Atlanticsalmon kidney sections with 3 different anti-PVCR antisera (anti-SalI,anti-4641, and anti-SKCaR) produces similar localizations of PVCRprotein within the tubules of salmon kidney. Staining produced byanti-SKCaR antiserum is identical to that produced by anti-4641antiserum, an anti-peptide antisera corresponding to extracellulardomain of mammalian PVCRs that is very similar to SKCaR (SEQ ID NO: 2).These PVCR protein patterns stained identically to that produced bySAL-1 antiserum. Anti-Sal-1 antiserum also exhibits a similar stainingpattern for the distribution of intestinal PVCR protein, as compared toanti-SKCaR. Thus, this new antiserum is specific for a PVCR in AtlanticSalmon tissues. This antiserum can be used to determine the presence,absence or amount of PVCR in various tissues of fish, using the methodsdescribed herein.

The Sal I antiserum is also useful in localization of SalmoKCaR proteinsin larval Atlantic salmon (See FIG. 37B). The Sal I antiserum localizesSalmoKCaR proteins in the developing nasal lamellae of anadromous fish,including Atlantic salmon and trout, skin, myosepta, otolith and sensoryepithelium. The myoseptae are collagenous sheets that separate thevarious muscle bundles in the fish. Myosepta are important in both thedevelopment of muscle in larval fish as well as its function for muscleforce generation in adult fish. Myosepta are also of significantcommercial importance since they are one of the principal determinantsof texture of smoked Atlantic salmon fillets.

The otolith is also of considerable importance to Atlantic salmon. It isa calcified structure located in the inner ear of salmon where it isclosely associated with epithelial cells responsible for sensing soundand direction. It is likely that the SalmoKCaRs associated with theotolith participate in the calcification of the otolith structure thatconsists of proteins and calcium precipitate.

A second peptide sequence was used for antibody production: Peptide #2:CSDDEYGRPGIEKFEKEM. (SEQ ID NO: 27)

This peptide was synthesized, derivatized in a manner identical to thatdescribed for Peptide #1 and antiserum was raised in rabbits asdescribed above. As expected, this antiserum (Salmo ADD) produced apattern of immonostaining on sections of juvenile Atlantic salmon thatis identical to that exhibited by Sal I. (See FIG. 37C). Since bothSalmoKCaR #1 and #2 but not SalmoKCaR #3 possess the carboxyl terminalsequence recognized by the Sal I antibody, the antibody-staining patterndisplayed by Sal I show the distribution of SalmoKCaR proteins #1 and #2but not #3 within the kidney of Atlantic salmon.

In contrast, the Salmo ADD antibody binds to a peptide sequence presentin the extracellular domain of all 3 SalmoKCaR proteins. Thus, any cellsthat possess no staining of Sal I but staining with Salmo ADD likelyexpress either SalmoKCaR #3 or some similar SalmoKCaR protein.

EXAMPLE 12 Use of Reverse Transcriptase Polymerase Chain Reaction(RT-PCR) to Detect Expression of PVCRs in Various Tissues

In Example 4, 2 degenerate primers, dSK-F3 (SEQ ID NO: 13) and dSK-R4(SEQ ID NO: 14), are disclosed. These two primers were used to amplifygenomic DNA and obtain the sequence of a portion of the genomic DNAsequences of PVCRs from various anadromous fish. These same primers canalso be used to amplify a portion of corresponding PVCR mRNA transcriptsin various tissues. DNA sequence analyses of amplified cDNAs fromspecific Atlantic salmon tissues (olfactory lamellae, kidney, urinarybladder) verifies these are all identical to certain genomic PVCRsequences described herein. These data show that:

1. PVCR mRNA transcripts are actually expressed in specific tissues ofanadromous fish. These data reinforce the data regarding PVCR proteinexpression as detected by anti-PVCR antisera.

2. RT-PCR methods can be used to detect and quantify the degree of PVCRexpression in various tissues, as a means to predict the readiness ofanadromous fish for transfer to seawater.

3. cDNA probes can be generated from specific tissues of anadromous fishfor use as specific DNA probes to either detect PVCR expression usingsolution or solid phase DNA-DNA or DNA-RNA nucleic acid hybridization orobtain putative PVCR protein sequences used for generation of specificanti-PVCR antisera.

RT-PCR Method:

Total RNA was purified from selected tissues using Teltest B reagent(Friendswood, Tex.) and accompanying standard protocol. A total of 5micrograms of total RNA was reverse transcribed with oligo dT primersusing Invitrogen's cDNA Cycle Kit (Invitrogen Inc, Madison, Wis.). Theresulting cDNA product was denatured and a second round of purificationwas performed. Two microliters of the resulting reaction mixture wasamplified in a PCR reaction (30 cycles of 1 min. @ 94° C., 2 min. @ 57°C., 3 min. @72° C.) using degenerate primers dSK-F3 (SEQ ID NO: 13) anddSK-R4 (SEQ ID NO: 14). The resulting products were electrophoresed on a2% (w/v) agarose gel using TAE buffer containing ethidium bromide fordetection of amplified cDNA products. Gels were photographed usingstandard laboratory methods.

DNA Sequencing of RT-PCR Products were Performed as Follows:

A total of 15 microliters of Atlantic Salmon urinary bladder, kidney andnasal lamellae RT-PCR reactions were diluted in 40 microliters of waterand purified by size exclusion on Amersham's MicroSpin S-400 HR spincolumns (Amersham Inc, Piscataway, N.J.). Purified DNA was sequencedusing degenerate PVCR primers (SEQ ID NO.: 13 and 14) as sequencingprimers. Automated sequencing was performed using an Applied BiosystemsInc. Model 373A Automated DNA Sequencer (University of Maine, Orono,Me.). The resulting DNA sequences were aligned using MacVector (GCG) andLaserGene (DNA STAR) sequence analysis software.

Detection of Amplified RT-PCR cDNA Products by Southern Blotting:

Alternatively, the presence of amplified PVCR products was detected bySouthern blotting analyses of gel fractionated RT-PCR products using a³²P-labeled 653 bp Atlantic salmon amplified genomic PCR product. Atotal of 10 microliters of each PCR reaction was electrophoresed on a 2%agarose gel using TAE buffer then blotted onto Magnagraph membrane(Osmonics, Westboro, Mass.). After crosslinking of the DNA, blots wereprehybridized and then probed overnight (68° C. in 6×SSC, 5× Denhardt'sReagent, 0.5% SDS, 100 ug/ml calf thymus DNA) with the 653 bp Atlanticsalmon PCR product (labeled with RadPrime DNA Labeling System, GibcoLife Sciences). Blots were then washed with 0.1×SSC, 0.1% SDS @ 55° C.and subjected to autoradiography under standard conditions.

FIG. 34 shows the results of RT-PCR amplification of a partial PVCR mRNAtranscript from various tissues of juvenile Atlantic salmon. RT-PCRreactions were separated by gel electrophoresis and either stained inethidium bromide(EtBr) or transferred to a membrane and Southern blottedusing a ³²P-labeled 653 bp genomic DNA fragment from the Atlantic salmonPVCR gene. FIG. 34 shows the detection of the PVCR in several tissuetypes of Atlantic Salmon using the RT-PCR method, as described herein.The types of tissue are gill, nasal lamellae, urinary bladder, kidney,intestine, stomach, liver, and brain.

EXAMPLE 13 Presence and Function of PVCR Protein in Nasal Lamellae andOlfactory Bulb as well as GI Tract of Fish

The data described herein described the roles of PVCR proteins in theolfactory organs (nasal lamellae and olfactory bulb) of fish as itrelates to the ability of fish to sense or “smell” both alterations inthe water salinity and/or ionic composition as well as specific aminoacids. These data are particularly applicable to anadromous fish(salmon, trout and char) that are either transferred from freshwaterdirectly to seawater or exposed to Process I or Process II in freshwaterand then transferred to seawater.

These data described herein were derived from a combination of sourcesincluding immunocytochemistry using anti-PVCR antisera, RT-PCRamplification of PVCRs from nasal lamellae tissue, studies of thefunction of recombinant aquatic PVCR proteins expressed in culturedcells where these proteins “sense” specific ions or amino acids as wellas electrophysiological recordings of nerve cell electrical activityfrom olfactory nerves or bulb of freshwater salmon.

The combination of immunocytochemistry and RT-PCR data, describedherein, reveal the presence of PVCR proteins in both major families offish (elasmobranch-shark; teleost-salmon) in both larval, juvenile andadult life stages. Immunocytochemistry analyses reveal that one or morePVCR proteins are present both on portions of olfactory receptor cellslocated in the nasal lamellae of fish (where they are bathed in waterfrom the surrounding environment) as well as on nerve cells that composeolfactory glomeruli present in the olfactory bulb of fish brain (wherethese cells are exposed to the internal ionic environment of the fish'sbody). Thus, from these locations fish are able to compare the ioniccomposition of the surrounding water with reference to their owninternal ionic composition. Alterations in the expression and/orsensitivity of PVCR proteins provides the means to enable fish todetermine on a continuous basis whether the water composition theyencounter is different from that they have been adapted to or exposed topreviously. This system is likely to be integral to both the control ofthe homing of salmon from freshwater to seawater as smolt and theirreturn to freshwater from seawater as adults. Thus, fish have theability to “smell” changes in water salinity directly via PVCR proteinsand respond appropriately to regulate remain in environments that arebest for their survival in nature.

One feature of this biological system is alteration in the sensitivityof the PVCR protein for divalent cations such as Ca²⁺ and Mg²⁺ bychanges in the NaCl concentration of the water. Thus, PVCRs in fisholfactory organs have different apparent sensitivity to Ca²⁺ in eitherthe presence or absence of NaCl. These data presented here are the firstdirect evidence for these functions via PVCR proteins present in theolfactory apparatus of fish.

Another feature of PVCR protein function in the olfactory apparatus offish is to modulate responses of olfactory cells to specific odorants(attractants or repellants). Transduction of cellular signals resultingfrom the binding of specific odorants to olfactory cells occurs viachanges in standing ionic gradients across the plasma membranes of thesecells. The binding of specific odorants to olfactory cells results inelectrical nerve conduction signals that can be recorded usingstandardized electrophysiological electrodes and equipment. Using thisapparatus, the olfactory apparatus of freshwater adapted salmon:

-   -   1. responded to PVCR agonists in a concentration-dependent        manner similar to that shown previously for other fish tissues        including that shown for winter flounder urinary bladder. These        data provide the functional evidence of the presence of a PVCR        protein; and    -   2. that the presence of a PVCR agonist reduces or ablates the        signal resulting from odorants including both attractants or        repellants. Thus, PVCRs in the olfactory apparatus of salmon        possess the capacity of modulating responses to various        odorants.

Another feature of PVCR proteins is their ability to “sense” specificamino acids present in surrounding environment. Using the full-lengthrecombinant SKCaR cDNA, functional SKCaR protein was expressed in HEKcells and shown to respond in a concentration-dependent manner to bothsingle and mixtures of L-amino acids. Since PVCR agonists includingamino acids as well as polyamines (putrescine, spermine and spermidine)are attractants to marine organisms including fish and crustaceans,these data provide for another means by which PVCR proteins would servenot only as modulators of olfaction in fish but also as sensors of aminoacids and polyamines themselves. PVCR proteins in other organs of fishincluding G.I. tract and endocrine organs of fish also function to sensespecific concentrations of amino acids providing for integration of awide variety of cellular processes in epithelial cells (amino acidtransport, growth, ion transport, motility and growth) with digestionand utilization of nutrients in fish.

Description of Experimental Results and Data Interpretation:

PVCR protein and mRNA are localized to the olfactory lamellae, olfactorynerve and olfactory bulb of freshwater adapted larval, juvenile andadult Atlantic salmon as well as the olfactory lamellae of dogfishshark:

FIG. 35 show representative immunocytochemistry photographs of PVCRprotein localization in olfactory bulb and nerve as well as olfactorylamellae in juvenile Atlantic salmon. The specificity of staining forPVCR protein is verified by the use of 2 distinct antisera each directedto a different region of the PVCR protein. Thus, antiserum anti-4641(recognizing an extracellular domain PVCR region) and antiserumanti-SKCaR (recognizing an intracellular domain PVCR region) exhibitsimilar staining patterns that include various glomeruli on serialsections of olfactory bulb. Using anti-SKCaR antiserum, specificstaining of PVCR proteins is observed in discrete regions of theolfactory nerve as well as epithelial cells in the nasal lamellae thatare exposed to the external ionic environment.

The presence of PVCR protein in both nasal lamellae cells as well asolfactory bulb and nerve shows that these respective PVCR proteins wouldbe able to sense both the internal and external ionic environments ofthe salmon. For this purpose, cells containing internally-exposed PVCRsare connected to externally-exposed PVCRs via electrical connectionswithin the nervous system. As shown schematically in FIG. 36, these datasuggest that externally and internally-exposed PVCRs function togetherto provide for the ability to sense the ionic concentrations of thesurrounding ionic environment using as a reference the ionicconcentration of the salmon's body fluids. Changes in the expressionand/or sensitivity of the external set of PVCRs vs internal PVCRs wouldthen provide a long term “memory” of the adaptational state of the fishas it travels through ionic environments of different composition. FIG.37 shows immunocytochemistry using anti-SKCaR antiserum that reveals thepresence of PVCR protein in both the developing nasal lamellae cells andolfactory bulb of larval Atlantic salmon only days after hatching (yolksac stage). As described herein, imprinting of salmon early indevelopment as well as during smoltification have been shown to be keyintervals in the successful return of wild salmon to their natal stream.The Sal I antiserum also localizes SalmoKCaR proteins in a variety oftissues in larval Atlantic salmon (FIG. 37B). These tissues include thedeveloping nasal lamellae of salmon and trout, their skin, myosepta,otolith and sensory epithelium. Myosepta are important in both thedevelopment of muscle in larval fish since they separate and define themuscle bundles of the salmon. Myosepta are also of significantcommercial importance since they are one of the principal determinantsof texture for smoked Atlantic salmon fillets. SalmoKCaR proteins arealso present in the otolith which is a calcified structure located inthe inner ear of the salmon where it is closely associated withepithelial cells responsible for sensing sound and direction. Thepresence of PVCR proteins at these developmental stages of salmonlifecycle indicate that PVCRs participate in this process.

Data obtained from using anti-SKCaR antiserum from other fish speciesincluding elasmobranchs display similar staining of PVCR protein incells (marked A) their nasal lamellae (FIG. 38). Use of othermethodology including RT-PCR using specific degenerate primers (FIG. 39)and ELISA methods (FIG. 40) detects the presence of PVCR proteins andmRNA in nasal lamellae of fish. While neither of these 2 techniquesprovide quantitative measurements as described, both sets of data areconsistent and show abundant PVCR protein present in this tissue.

Measurement of Extracellular Electrical Potentials (EEG's) fromOlfactory Nerve from Freshwater Adapted Atlantic Salmon Reveals thePresence of Functional PVCR Proteins:

FIG. 41 displays representative recordings obtained from 6 freshwateradapted juvenile Atlantic salmon (approximately 300-400 gm) usingmethods similar to those described in Bodznick, D. J Calcium ion: anodorant for natural water discriminations and the migratory behavior ofsockeye salmon, Comp. Physiol. A 127:157-166 (1975), and Hubbard, P C,et al., Olfactory sensitivity to changes in environmental Ca2+ in themarine teleost Sparus Aurata, J. Exp. Biol. 203:3821-3829 (2000). Afteranaesthetizing the fish, it was placed in V-clamp apparatus where itsgills were irrigated continuously with aerated seawater and its nasallamellae bathed continuously by a stream of distilled water via a tubeheld in position in the inhalant olfactory opening. The olfactory nervesof the fish were exposed by removal of overlying bony structures.Stimuli were delivered as boluses to the olfactory epithelium via a 3way valve where 1 cc of water containing the stimulus was rapidlyinjected into the tube containing a continuously stream of distilledwater. Extracellular recordings were obtained using high resistancetungsten electrodes where the resultant amplified analog signals (GrassAmplifier Apparatus) were digitized, displayed and analyzed by computerusing MacScope software. Using this experimental approach, stable andreproducible recordings could be obtained for up to 6 hr after theinitial surgery on the fish.

As shown in FIG. 41, irrigation of salmon olfactory epithelium withdistilled water produces minimal generation of large signals inolfactory nerve. The data in FIG. 41 are displayed as both rawrecordings (left column) and the corresponding integrated signals foreach raw recording shown in the right column. Exposure of the olfactoryepithelium to 500 micromolar L-alanine (a well known amino acidattractant for fish) produces large increases in both the firingfrequency and amplitude in the olfactory nerve lasting approximately 2seconds in duration. Similarly, application of either 1 mM Ca²⁺ or 250mM NaCl also produce responses in EEG activity. To test for the presenceof functional PVCR protein, the olfactory epithelium was exposed to 50micromolar gadolinium (Gd³⁺-a PVCR agonist) and also obtained aresponse. FIG. 42 shows dose response data from multiple fish to variousPVCR agonists or modulators where the relative magnitudes of individualolfactory nerve response were normalized relative to the responseproduced by the exposure of the olfactory epithelium to 10 mM Ca²⁺. Asshown in FIG. 42, the olfactory epithelium of freshwater adaptedjuvenile salmon is very sensitive to Ca²⁺ where the half maximalexcitatory response (EC₅₀) is approximately 1-10 micromolar. Similarly,exposure of olfactory epithelium to the PVCR agonist Gd³⁺ producesresponses of a similar magnitude to those evoked by Ca²⁺ in aconcentration range of 1-10 micromolar. In contrast, olfactoryepithelium responses to Mg²⁺ do not occur until 10-100 micromolarsolutions are applied. These dose response curves (EC₅₀ Gd⁺³≦Ca²⁺<Mg²⁺)are similar to those obtained for PVCR modulated responses in other fishepithelium (flounder urinary bladder NaCl-mediated water transport-seeSKCaR application).

In contrast, analysis of the olfactory epithelium responses to NaClexposure shows that it is unresponsive until a concentration of 250millimolar NaCl is applied. Since NaCl does not directly activate PVCRsin a manner such as Gd⁺³ Ca²⁺ or Mg²⁺ but rather reduces the sensitivityof PVCRs to these agonists, these data are also consistent with thepresence of an olfactory epithelium PVCR. The response evoked byexposure of the epithelium to significant concentrations of NaCl likelyoccurs via other PVCR independent mechanisms.

These data suggest that PVCR proteins present in olfactory epitheliumare capable of sensing and generating corresponding olfactory nervesignals in response to PVCR agonists at appropriate concentrations indistilled water.

Addition of PVCR agonists such as Ca2+ or Gd3+ to distilled watercontaining well known salmon repellants reversibly ablates the responseof the olfactory epithelium to these stimuli:

FIG. 43 shows representative data obtained from a single continuousrecording where the olfactory epithelium was first exposed to awell-known repellant, mammalian finger rinse. Finger rinse is obtainedby simply rinsing human fingers of adherent oils and fatty acids usingdistilled water and has been shown previously to be a powerful repellantstimulus both in EEG recordings as well as behavioral avoidance assays(Royce-Malmgren and W. H Watson J. Chem. Ecology 13:533-546 (1987)).Note however that inclusion of the PVCR agonists 5 mM Ca²⁺ or 50micromolar Gd³⁺ reversibly ablated the response by the olfactoryepithelium to mammalian finger rinse. These data show that PVCR agonistsmodulated the response of the olfactory epithelium to an odorant such asmammalian finger rinse. The ablation of responses to both the PVCRagonists as shown in FIG. 42 as well as mammalian finger rinse indicatethat there are some complex interactions between PVCR proteins and otherodorant receptors. It is also extremely unlikely that inclusion of PVCRagonists removed all the stimulatory components of mammalian fingerrinse from solution such that they were not able to stimulate theepithelium.

Addition of PVCR agonists such as Ca2+ or Gd3+ but not NaCl to distilledwater containing the well known salmon attractant L-alanine reversiblyablates the response of the olfactory epithelium to these stimuli:

FIG. 44 shows a time series of stimuli (2 min between each stimulus in asingle fish) similar to that displayed on FIG. 43 except that 500micromolar L-Alanine (a salmon attractant) was used to produce a signalin the olfactory nerve. Note that the addition of either 5 mM Ca²⁺(recording #2) or 50 micromolar Gd³⁺ (recording #7) to 500 micromolarL-alanine resulted in the complete loss of the corresponding responsefrom the olfactory nerve after injection of this mixture. In both cases,this was not due to a permanent alteration of the olfactory epitheliumby either of these PVCR agonists because a subsequent identical stimuluswithout the PVCR agonist (recordings #3 and #8) caused a return of thesignal. It is noteworthy that in the case of Gd³+addition, the magnitudeof the subsequent L-alanine signal was decreased as compared to control(compare recordings #6 vs #8) indicating that the olfactory epitheliumprefers an interval of recovery from its exposure to this potent PVCRagonist. However, the alteration of response to the L-Alanine stimulusis not permanent or nonspecific since combining the same dose ofL-Alanine with 250 mM NaCl resulted initially in a similar response(recordings #4 and #9) followed by an enhanced response to L-Alaninealone (recordings #5 and #10).

In summary, the data displayed in FIGS. 43 and 44 show that inclusion ofa PVCR agonist in solutions containing either a repellant (finger rinse)or attractant (L-alanine) causes a dramatic reduction in the response ofthe olfactory epithelium to those odorants. For both repellants andattractants, some form of complex interactions occur within olfactoryepithelial cells since mixing of PVCR agonists and odorants renders theepithelia temporary unresponsive to either stimulus. While the nature ofsuch interactions are not known at the present time, such interactionsdo not occur at the level of the PVCR molecule itself as shown by datafrom experiments using recombinant PVCR protein SKCaR. As furtherdescribed herein, inclusion of amino acids in the presence of Ca²⁺enhances the response of SKCaR to ambient Ca²⁺ concentrations.Regardless of their nature, these negative modulatory effects of PVCRagonists including Ca²⁺ is likely to produce major effects on howfreshwater salmon smell objects in their environment after transfer froma low calcium to a high calcium environment. Use of this assay systemwould permit the identification and analyses of both specific classes ofPVCR agonists and antagonists as well as the specific effects of eachPVCR modulator on specific odorants including both repellants andattractants.

Recombinant PVCR protein SKCaR possesses the capability to senseconcentrations of amino acids after its expression in human embryonickidney (HEK) cells:

Full length recombinant dogfish (Squalus acanthias) shark kidney calciumreceptor (SKCaR) was expressed in human embryonic kidney cells usingmethods described herein. The ability of SKCaR to respond to individualamino acids as well as various mixtures was quantified using FURA-2ratio imaging fluorescence.

FIG. 45 shows a comparison of fluorescence tracings of FURA2-loadedcells stably expressing SKCaR that were bathed in physiological saline(125 mM NaCl, 4 mM KCl, 0.5 mM CaCl₂, 0.5 MgCl₂, 20 mM HEPES (NaOH),0.1% D-glucose pH 7.4) in the presence or absence of 10 mM L-Isoleucine(L-Ile) before being placed into the fluorimeter. Baseline extracellularCa²⁺ concentration was 0.5 mM. Aliquots of Ca²⁺ were added to producefinal extracellular concentrations of 2.5 mM, 5 mM, 7.5 mM, 10 mM and 20mM Ca²⁺ with changes in the fluorescence recorded. Note that increasesin cell fluorescence were greater in the presence of 10 mM Phe forextracellular Ca²⁺ concentrations less than 10 mM.

FIG. 46 shows data plotted from multiple experiments as described inFIG. 45 where the effects of 10 mM Phe, 10 mM Ile or an amino acidmixture (AA Mixture) containing all L-isomers in the followingconcentrations in micromoles/liter: 50 Phe, 50 Trp, 80 His, 60 Tyr, 30Cys, 300 Ala, 200 Thr, 50 Asn, 600 Gln, 125 Ser, 30 Glu, 250 Gly, 180Pro, 250 Val, 30 Met, 10 Asp, 200 Lys, 100 Arg, 75 Ile, 150 Leu. Notethat both 10 mM Phe and 10 mM Ile as well as the mixture of amino acidsincrease SKCaR's response to a given Ca²⁺ concentration. Thus, thesedata show that presence of amino acids either alone or in combinationincrease the apparent sensitivity to Ca²⁺ permitting SKCaR to “sense”amino acids in the presence of physiological concentrations of Ca²⁺.These data obtained for SKCAR are comparable to those obtained for thehuman CaR.

The significance of these data for aquatic organisms stand in markedcontrast to the roles of human CaRs amino acid sensing capabilities.FIG. 45 shows that SKCaR's maximal capability to sense amino acids isconfined to a range of Ca²⁺ that is present both in aquatic externalenvironments as well as the body fluids of various fish. The followingphysiological processes occur: 1) Sensing of amino acids in the proximalintestine and pyloric caeca of fish: The PVCR present on the apicalsurface of intestinal epithelial cells is capable of responding to aminoacids such as tryptophan as part of the Process II. Inclusion oftryptophan in the feed of fish interacts with the intestinal PVCR toimprove the development of juvenile anadromous fish to tolerate seawatertransfer. 2) In both adult, juvenile and larval fish, PVCR localized tothe apical membrane of stomach and intestinal epithelial cells could“sense” the presence of amino acids produced by the proteolysis ofproteins into amino acids. This mechanism could be used to inform bothepithelial and neuroendocrine cells of the intestine of the presence ofnutrients (proteins) and trigger a multitude of responses includinggrowth and differentiation of intestinal epithelia as well as theiraccompanying transport proteins, secretion or reabsorption of ions suchas gastric acid. The apical PVCR also regulates the secretion ofintestinal hormones such as cholecystokin (CCK) and others. 3) PVCRproteins present in cells of the nasal lamellae of fish “smell” bothwater salinity (via Ca²⁺, Mg²⁺ and NaCl) and amino acids which is anexample of an attractant. At the present time, it is unclear whether theamino acid sensing capabilities of PVCRs are utilized by the olfactoryepithelium to enable fish to smell various amino acid attractants.

These data show that PVCR sensing of amino acids occurs in a range ofextracellular calcium that is present in various concentrations ofseawater present in estuaries and fish migration routes as well asvarious compartments of a fish's body including serum and body cavitiesincluding intestine, pyloric caeca and kidney where transepithelialamino acid absorption occurs. These data constitute the first reportshowing the amino acid sensitivity of a PVCR in fish.

Companion Patent Application Nos. (not yet assigned; Attorney Docket No:2213.1006-005 and 2213.1006-007), both entitled “PolyvalentCation-sensing Receptor in Atlantic Salmon,” filed on Apr. 18, 2002;patent application Ser. No. 09/687,373, entitled “Growing Marine Fish inFresh Water,” filed on Oct. 12, 2000; PCT Application No.:PCT/US01/31625, entitled “Growing Marine Fish in Fresh Water,” filedOct. 11, 2001; patent application Ser. No. 09/687,476, entitled “Methodsfor Raising Pre-adult Anadromous Fish,” filed on Oct. 12, 2000; patentapplication Ser. No. 09/687,372, entitled “Methods for Raising Pre-adultAnadromous Fish,” filed on Oct. 12, 2000; patent application Ser. No.09/687,477, entitled “Methods for Raising Pre-adult Anadromous Fish,”filed on Oct. 12, 2000; patent application Ser. No. 09/975,553, entitled“Methods for Raising Pre-adult Anadromous Fish,” filed on Oct. 11, 2001;International PCT Application No. PCT/US01/31562, entitled, “Methods forRaising Pre-adult Anadromous Fish,” filed on Oct. 11, 2001; ProvisionalPatent Application No. 60/382,464, “Methods for Growing and ImprintingFish Using an Odorant,” filed Oct. 11, 2001; are all hereby incorporatedby reference in their entirety.

Additionally, U.S. Pat. No 6,334,391, issued on Jan. 8, 2002,International PCT application No. PCT/US97/05031, filed on Mar. 27,1997, and application Ser. No. 08/622,738 filed Mar. 27, 1996, allentitled, “Polycation Sensing Receptor in Aquatic Species and Methods ofUse Thereof” are all hereby incorporated by reference in their entirety.

All relevant portions of literature articles, references, patentapplications, patent publications, and patents cited herein are herebyincorporated by referenced in their entirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A probe that hybridizes under high stringency conditions to a nucleicacid molecule that comprises a nucleic acid sequence having SEQ ID NO:9; or the coding region of SEQ ID NO: 9; but not to SEQ ID NO: 1 or thecoding region of SEQ ID NO: 1 under said conditions.
 2. A probe having asequence from SEQ ID NO: 9, but not SEQ ID NO:
 1. 3. A nucleic acidprobe having a sequence from SEQ ID NO: 9, but not SEQ ID NO:
 1. 4. ADNA probe having a sequence from SEQ ID NO: 9, but not SEQ ID NO: 1.